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1.  Exploiting the downhill folding regime via experiment 
HFSP Journal  2008;2(6):342-353.
Traditionally, folding experiments have been directed at determining equilibrium and relaxation rate constants of proteins that fold with two-state-like kinetics. More recently, the combination of free energy surface approaches inspired by theory with the discovery of proteins that fold in the downhill regime has greatly widened the battlefield for experimentalists. Downhill folding proteins cross very small or no free energy barrier at all so that all relevant partially folded conformations become experimentally accessible. From these combined efforts we now have tools to estimate the height of thermodynamic and kinetic folding barriers. Procedures to measure with atomic resolution the structural heterogeneity of conformational ensembles at varying unfolding degrees are also available. Moreover, determining the dynamic modes driving folding and how they change as folding proceeds is finally at our fingertips. These developments allow us to address via experiment fundamental questions such as the origin of folding cooperativity, the relationship between structure and stability, or how to engineer folding barriers. Moreover, the level of detail attained in this new breed of experiments should provide powerful benchmarks for computer simulations of folding and force-field refinement.
PMCID: PMC2645577  PMID: 19436488
2.  A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations 
PLoS Computational Biology  2009;5(8):e1000452.
Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data.
Author Summary
Understanding the mechanism by which proteins find their folded state is a holy grail of computational biology. Accurate all-atom simulations have the potential to describe such a process in great detail, but, unfortunately, folding of most proteins takes place on a time scale that is still not accessible to routine computer simulations. We introduce here an approach that allows for constructing an accurate kinetic and thermodynamic model of folding (or other complex biological processes) using trajectories in which the process under investigation is forced to happen in a short simulation time by an appropriate external bias. An important strength of this approach is the possibility of identifying and characterizing misfolded conformations that, in some proteins, are related to important diseases. We use this method to study the folding of Trp-cage, predicting the structure of the folded state and the presence of several intermediates. We find that, surprisingly, fully unstructured “unfolded” states relax towards the folded conformation rather quickly. The slowest relaxation time of the system is instead related to the equilibration between the folded state and another compact structure that acts as a kinetic trap. Thus, the experimental folding time would be determined primarily by this process.
PMCID: PMC2711228  PMID: 19662155
3.  Unfolding Simulations Reveal the Mechanism of Extreme Unfolding Cooperativity in the Kinetically Stable α-Lytic Protease 
PLoS Computational Biology  2010;6(2):e1000689.
Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease α-lytic protease (αLP) has done just that, as its unfolding is both very slow (t1/2 ∼1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to αLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the αLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both αLP and trypsin. The simulated αLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that αLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of αLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases.
Author Summary
Proteins, synthesized as linear polymers of amino acids, fold up into compact native states, burying their hydrophobic amino acids into their interiors. Protein folding minimizes the non-specific interactions that unfolded protein chains can make, which include aggregation with other proteins and degradation by proteases. Unfortunately, even in the native state, proteins can partially unfold, opening up regions of their structure and making these adverse events possible. Some proteins, particularly those in harsh environments full of proteases, have evolved to virtually eliminate partial unfolding, significantly reducing their rate of degradation. This elimination of partial unfolding is termed “cooperative,” because unfolding is an all-or-none process. One class of proteins has diverged into two families, one bacterial and highly cooperative and the other animal and non-cooperative. We have used detailed simulations of unfolding for members of each family, α-lytic protease (bacterial) and trypsin (animal) to understand the unfolding pathways of each and the mechanism for the differential unfolding cooperativity. Our results explain prior biochemical experiments, reproduce the large difference in unfolding cooperativity between the families, and point to the interface between α-lytic protease's two domains as essential to establishing unfolding cooperativity. As seen in an unrelated protein family, generation of a cooperative domain interface may be a common evolutionary response for ensuring the highest protein stability.
PMCID: PMC2829044  PMID: 20195497
4.  How Kinetics within the Unfolded State Affects Protein Folding: an Analysis Based on Markov State Models and an Ultra-Long MD Trajectory 
The journal of physical chemistry. B  2013;117(42):10.1021/jp401962k.
Understanding how kinetics in the unfolded state affects protein folding is a fundamentally important yet less well-understood issue. Here we employ three different models to analyze the unfolded landscape and folding kinetics of the miniprotein Trp-cage. The first is a 208 μs explicit solvent molecular dynamics (MD) simulation from D. E. Shaw Research containing tens of folding events. The second is a Markov state model (MSM-MD) constructed from the same ultra-long MD simulation; MSM-MD can be used to generate thousands of folding events. The third is a Markov state model built from temperature replica exchange MD simulations in implicit solvent (MSM-REMD). All the models exhibit multiple folding pathways, and there is a good correspondence between the folding pathways from direct MD and those computed from the MSMs. The unfolded populations interconvert rapidly between extended and collapsed conformations on time scales ≤ 40 ns, compared with the folding time of ≈ 5 μs. The folding rates are independent of where the folding is initiated from within the unfolded ensemble. About 90 % of the unfolded states are sampled within the first 40 μs of the ultra-long MD trajectory, which on average explores ~27 % of the unfolded state ensemble between consecutive folding events. We clustered the folding pathways according to structural similarity into “tubes”, and kinetically partitioned the unfolded state into populations that fold along different tubes. From our analysis of the simulations and a simple kinetic model, we find that when the mixing within the unfolded state is comparable to or faster than folding, the folding waiting times for all the folding tubes are similar and the folding kinetics is essentially single exponential despite the presence of heterogeneous folding paths with non-uniform barriers. When the mixing is much slower than folding, different unfolded populations fold independently leading to non-exponential kinetics. A kinetic partition of the Trp-cage unfolded state is constructed which reveals that different unfolded populations have almost the same probability to fold along any of the multiple folding paths. We are investigating whether the results for the kinetics in the unfolded state of the twenty-residue Trp-cage is representative of larger single domain proteins.
PMCID: PMC3808496  PMID: 23705683
Protein Folding; Unfolded State Kinetics
5.  Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins 
eLife  2014;3:e03348.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.
eLife digest
Many processes in living things need molecules to be transported within, or between, cells. For example, damaged or waste molecules are transported within a cell to structures that can break the molecules down, while nerve impulses are transmitted from one neuron to the next via the release of signaling molecules.
Cells—and the compartments within cells—are surrounded by membranes that act as barriers to certain molecules. Vesicles are small, membrane-enclosed packages that are used to transport molecules between different membranes; and in order to release its cargo, a vesicle must fuse with its target membrane. To fuse like this, the forces that act to push membranes away from one another need to be overcome. Proteins called SNARES, which are embedded in both membranes, are the molecular engines that power the fusion process. Once the SNARE proteins from the vesicle and the target membrane bind, they assemble into a more compact complex that pulls the two membranes close together and allows fusion to take place.
The final shape of an assembled SNARE complex is essentially the same for all SNARE complexes; however, it is not known whether all of these complexes fold using the same method. Now Zorman et al. have used optical tweezers—an instrument that uses a highly focused laser beam to hold and manipulate microscopic objects—to observe the folding and unfolding of four different types of SNARE complex. All four SNARE complexes followed the same step-by-step process: the leading ends of the SNARE proteins slowly bound to each other; the process paused; then the rest of the proteins rapidly ‘zippered’ together.
Zorman et al. revealed that, although the steps in the processes were the same, the energy released in the last step was different when different complexes assembled. This suggests that the energy released by the ‘zippering’ of different SNARE proteins is optimized to match the required speed of different membrane fusion events. Furthermore, Zorman et al. propose that the reason why the majority of energy is released in the later stages of complex assembly is because this is when the repulsion between the two membranes is strongest.
The discoveries of Zorman et al. will now aid future efforts aimed at understanding better how the numerous other proteins that interact with SNARE proteins regulate the process of membrane fusion.
PMCID: PMC4166003  PMID: 25180101
SNAREs; optical tweezers; protein folding; membrane fusion; SNARE assembly; energy landscape; E. coli
6.  Evolutionary fates within a microbial population highlight an essential role for protein folding during natural selection 
Physicochemical properties of molecules can be linked directly to evolutionary fates of a population in a quantitative and predictive manner.Reversible- and irreversible-folding pathways must be accounted for to accurately determine in vitro kinetic parameters (KM and kcat) at temperatures or conditions in which a significant fraction of free enzyme is unfolded.In vivo population dynamics can be reproduced using in vitro physicochemical measurements within a model that imposes an activity threshold above which there is no added fitness benefit.
In nature, evolution occurs through the continuous adaptation of a population to its environment. The success or failure of organisms during adaptation is based on changes in molecular structure that give rise to changes in fitness that dictate evolutionary fates within a population. Although the conceptual link between genotype, phenotype, and fitness is clear, the ability to relate these complex adaptive landscapes in a quantitative manner remains difficult (Kacser and Burns, 1981; Dykhuizen et al, 1987; Weinreich et al, 2006). Dean and Thornton (2007) coined the term ‘functional synthesis' to capture the synergy between evolutionary and molecular biology to address important questions such as the evolution of complexity. The ‘functional synthesis,' in its most fully realized form, is an integrated systems biology approach to evolutionary dynamics that links physicochemical properties of molecules to evolutionary fates in a quantitative and predictive manner.
Functional synthesis flourishes in an experimental framework that allows investigators to directly link population dynamics (fitness) to changes in molecular function that result from alterations at the nucleotide level. The ‘weak link' approach was developed to tightly couple adaptive changes within the genome to changes in fitness and provide a population-based approach that can be used to examine alterations in function and fitness at the level of atomic structure and function (Counago and Shamoo, 2005; Counago et al, 2006). A homologous recombination strategy was used to replace the chromosomal copy of the essential adenylate kinase gene (adk) of the thermophilic bacterium Geobacillus stearothermophilus with that of the mesophile Bacillus subtilis. Recombinant G. stearothermophilus cells that expressed only B. subtilis adenylate kinase (AKBSUB) were unable to grow at temperatures higher than 55°C because of heat inactivation of the mesophilic enzyme and consequent disruption of adenylate homeostasis (Counago and Shamoo, 2005). Continuously growing populations of bacteria were then subjected to selection at increasing temperatures (from 55 to 70°C) that favor changes in the one gene not adapted for thermostability, adk. During the course of selection, the population was sampled and intermediates of adaptation were observed as mutations to adk. The first mutant to reach fixation was a single mutation AKBSUB Q199R (the glutamine at position 199 replaced with arginine). AKBSUB Q199R was eventually replaced at 62–63°C by five double mutants that arose nearly simultaneously within the population and share AKBSUB Q199R as their progenitor (Figure 4C). Changes to AK activity and thermal stability that resulted from mutation had direct consequences for cellular fitness and, therefore, met our goal for an experimental system that allows us to develop and test models for quantitative molecular evolution. These enzyme activities and stabilities were examined to determine how the mutant populations traversed the adaptive landscape to increased fitness (Counago et al, 2006).
We found that reversible- and irreversible-folding pathways as well as a ‘physiological threshold' above which fitness changes are minimal are necessary to reproduce the in vivo evolutionary fates of the population. Protein-folding parameters must be accounted for to accurately determine in vitro kinetic parameters (KM and kcat) at temperatures in which a significant fraction of free enzyme is unfolded (Scheme I and Equation 1).
Scheme I
Thermostability was assayed using differential scanning calorimetry (DSC) (Figure 4A) and the fraction of unfolded protein (YU) was then extended to accurately predict the extent of stabilization, shift in Tm, in the presence of ligand. The kinetic parameters determined at specific temperatures were then used to construct a temperature-dependent formulation of Equation (1) to model in vitro activity at any given ATP concentration and any temperature (Figure 4B).
Here, we have modeled fitness as a function of in vitro enzyme activity, which is a product of both activity and stability, and the application of a threshold that provides an upper limit on fitness. We hypothesize that an activity threshold exists above which no added fitness benefit is attained (the ‘physiological threshold'). However, as activity falls below this threshold, AK becomes rate limiting and fitness is negatively affected. The experimentally observed rise and fall of mutant alleles is shown in Figure 4C, whereas those predicted from our in vitro model are shown as Figure 4D. This model can successfully reproduce frequencies of mutants in a polymorphic population, including the transient success of three minor mutants and order of disappearance from the population, given only in vitro data and allowing for the activity threshold to be fit to the observed outcomes (Figure 4D). An appealing aspect of our fitness function is that it permits an evaluation of specific and quantitative aspects of protein stability and activity relative to evolutionary fates.
In vivo, diversity within a population is generated by a variety of mechanisms that span single nucleotide changes to genome-wide rearrangements and horizontal gene transfer. However, changes are generated within an organism, it is the physicochemical characteristics of the resulting macromolecules and their resultant changes in the fitness of the organism that are the ‘grist for the mill' of natural selection. Recent work has shown that adaptability can be facilitated by the accumulation of near neutral or even modestly destabilizing mutations that provide more possibilities for success. Chaperones have an important function in buffering biological systems against these destabilizing mutations as well as mistakes in translation that lead to polymorphic populations and have been shown to increase rates of adaptation (Rutherford, 2003; Drummond and Wilke, 2008; Tokuriki and Tawfik, 2009a). Thus, adaptation through protein evolution is circumscribed by protein stability. As most mutational events will be destabilizing (Tokuriki and Tawfik, 2009b), higher mutation rates can lead to decreases in fitness eventually leading to extinction (Zeldovich et al, 2007; Chen and Shakhnovich, 2009). Although our system links the physicochemical properties of adaptive changes that increase stability, the principles apply equally to those changes that might decrease stability of the ensemble either through mutation or translational errors (Drummond and Wilke, 2008). Thus, regardless of how protein diversity is generated, evolutionary dynamics will likely be strongly coupled to stability and function.
Systems biology can offer a great deal of insight into evolution by quantitatively linking complex properties such as protein structure, folding, and function to the fitness of an organism. Although the link between diseases such as Alzheimer's and misfolding is well appreciated, directly showing the importance of protein folding to success in evolution has been more difficult. We show here that predicting success during adaptation can depend critically on enzyme kinetic and folding models. We used a ‘weak link' method to favor mutations to an essential, but maladapted, adenylate kinase gene within a microbial population that resulted in the identification of five mutants that arose nearly simultaneously and competed for success. Physicochemical characterization of these mutants showed that, although steady-state enzyme activity is important, success within the population is critically dependent on resistance to denaturation and aggregation. A fitness function based on in vitro measurements of enzyme activity, reversible and irreversible unfolding, and the physiological context reproduces in vivo evolutionary fates in the population linking organismal adaptation to its physical basis.
PMCID: PMC2925523  PMID: 20631681
adenylate kinase; enzyme kinetics; experimental evolution; fitness functions; protein folding
7.  The Proteostasis Boundary in Misfolding Diseases of Membrane Traffic 
FEBS letters  2009;583(16):2639-2646.
Protein function is regulated by the proteostasis network (PN) (Balch et al. (2008) Science, 319:916), an integrated biological system that generates and protects the protein fold. The composition of the PN is regulated by signaling pathways including the unfolded protein response (UPR), the heat shock response (HSR), the ubiquitin proteasome system (UPS) and epigenetic programs. Mismanagement of protein folding and function during membrane trafficking through the exocytic and endocytic pathways of eukaryotic cells by the PN is responsible for a wide range of diseases that include, among others, lysosomal storage diseases, myelination diseases, cystic fibrosis, systemic amyloidoses such as light chain myeloma, and neurodegenerative diseases including Alzheimer’s. Toxicity from misfolding can be cell autonomous (affect the producing cell) or cell non-autonomous (affect a non-producing cell) or both, and have either a loss-of-function or gain-of-toxic function phenotype. Herein, we review the role of the PN and its regulatory transcriptional circuitry likely to be operational in managing the protein fold and function during membrane trafficking. We emphasize the enabling principle of a ‘proteostasis boundary (PB)’ (Evans et al. (2009) Ann. Rev. Biochem. In press, Epub). The PB is defined by the combined effects of the kinetics and thermodynamics of folding and the kinetics of misfolding, which are linked to the variable and adjustable PN capacity found different cell types. Differences in the PN account for the versatility of protein folding and function in health, and the cellular and tissue response to mutation and environmental challenges in disease. We discuss how manipulation of the folding energetics or the PB through metabolites and pharmacological intervention provides multiple routes for restoration of biological function in trafficking disease affecting human health.
PMCID: PMC2805282  PMID: 19708088
8.  Antigenic Fingerprinting of H5N1 Avian Influenza Using Convalescent Sera and Monoclonal Antibodies Reveals Potential Vaccine and Diagnostic Targets 
PLoS Medicine  2009;6(4):e1000049.
Using whole-genome-fragment phage display libraries, Hana Golding and colleagues identify the viral epitopes recognized by serum antibodies in humans who have recovered from infection with H5N1 avian influenza.
Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics.
Methods and Findings
To address this need, we generated whole-genome–fragment phage display libraries (GFPDL) expressing fragments of 15–350 amino acids covering all the proteins of A/Vietnam/1203/2004 (H5N1). These GFPDL were used to analyze neutralizing human monoclonal antibodies and sera of five individuals who had recovered from H5N1 infection. This approach led to the mapping of two broadly neutralizing human monoclonal antibodies with conformation-dependent epitopes. In H5N1 convalescent sera, we have identified several potentially protective H5N1-specific human antibody epitopes in H5 HA[(-10)-223], neuraminidase catalytic site, and M2 ectodomain. In addition, for the first time to our knowledge in humans, we identified strong reactivity against PB1-F2, a putative virulence factor, following H5N1 infection. Importantly, novel epitopes were identified, which were recognized by H5N1-convalescent sera but did not react with sera from control individuals (H5N1 naïve, H1N1 or H3N2 seropositive).
This is the first study, to our knowledge, describing the complete antibody repertoire following H5N1 infection. Collectively, these data will contribute to rational vaccine design and new H5N1-specific serodiagnostic surveillance tools.
Editors' Summary
Every winter, millions of people catch influenza, a viral infection of the airways. Most recover quickly but seasonal influenza outbreaks (epidemics) kill about half a million people annually. These epidemics occur because small but frequent changes in the viral proteins (antigens) to which the human immune system responds mean that an immune response produced one year by infection or through vaccination provides only partial protection against influenza the next year. Influenza viruses also occasionally appear that contain major antigenic changes. Human populations have little or no immunity to such viruses (which often originate in animals or birds), so they can start deadly global epidemics (pandemics ). Worryingly, the last influenza pandemic occurred in 1968 and many experts fear that another pandemic is now overdue. The trigger for such a pandemic, they think, could be the avian (bird) H5N1 influenza virus, which first appeared in 1996 in a goose in China. The name indicates the types of two major influenza antigens present in the virus: H5N1 carries type 5 hemagglutinin and type 1 neuraminidase.
Why Was This Study Done?
H5N1 has caused about 400 confirmed cases of human influenza and more than 250 deaths in the past decade but it has not started a human pandemic because it cannot pass easily between people. However, it could possibly acquire this ability at any time, so it is a priority to develop both vaccines that will provide protection against a pandemic H5N1 viral strain, as well as antibody-based antiviral therapies for people not protected by vaccination (antibodies are proteins produced by the immune system that help to fight infections; people can sometimes be protected from infection by injecting them with pre-prepared antibodies). To do this, scientists need to know how the human immune system responds to the H5N1 virus. In particular, they need to know which parts of the virus the immune system can detect and make antibodies against. In this study, therefore, the researchers characterize the specific antibody responses found in people recovering from infection with H5N1.
What Did the Researchers Do and Find?
The researchers made several “genome-fragment phage display libraries”, collections of bacterial viruses (phages) engineered so that each phage makes one of many possible short pieces (polypeptides) of a nonphage protein. Such “libraries” can be used to investigate which fragments are recognized by antibodies from a given source. In this case, several libraries were made that contained fragments of the genome of the H5N1 strain responsible for an outbreak of human influenza in Vietnam in 2004–2005 (A/Vietnam/1203/2004). The researchers used these libraries to analyze the antibodies made by five Vietnamese people recovering from infection with A/Vietnam/1203/2004. H5N1 convalescent blood samples, the researchers report, contained antibodies that recognized small regions (“epitopes”) in several viral proteins, including hemagglutinin, neuraminidase, a structural protein called M2, and a viral protein called PB1-F2 that is partly responsible for the severity of H5N1 infections. Several of the novel epitopes identified were not recognized by antibodies in blood taken from people recovering from infection with other influenza viruses. The researchers also used their phage display libraries to analyze two neutralizing human monoclonal antibodies generated from patients infected with A/Vietnam/1203/2004 (neutralizing antibodies protect mice against normally lethal challenge with H5N1; monoclonal antibodies are generated in the laboratory by creating continuously growing cell lines that produce a single type of antibody). Importantly, both of the neutralizing monoclonal antibodies recognized “noncontinuous conformation-dependent epitopes”—protein sequences that are not adjacent to one another in the polypeptide sequence of the protein, but that lie close together in space because of the way the protein is folded up.
What Do These Findings Mean?
Although some aspects of the antibody repertoire produced in people exposed to the H5N1 influenza virus may have been missed in this analysis, these findings provide important and detailed new information about how the human immune system responds to infection with this virus. In particular, they show that people recovering from H5N1 infection make a diverse range of antibodies against several viral proteins for at least six months and identify specific parts of H5N1 that may be particularly good at stimulating a protective immune response. This information can now be used to help design vaccines against H5N1 and antibody-based therapies for the treatment of H5N1 infections, and to develop new tools for monitoring outbreaks of avian influenza in human populations.
Additional Information
Please access these Web sites via the online version of this summary at
This study is further discussed in a PLoS Medicine Perspective by Malik Peiris
The US Centers for Disease Control and Prevention provides information for about influenza for patients and professionals, including specific information on avian and pandemic influenza (in several languages)
The World Health Organization provides information on influenza (in several languages) and on H5N1 avian influenza (in several languages), and a global timeline about H5N1 avian influenza infection in birds and people
The UK Health Protection Agency provides information on avian, pandemic, and epidemic (seasonal) influenza
MedlinePlus provides a list of links to other information about influenza and bird flu (in English and Spanish)
PMCID: PMC2661249  PMID: 19381279
9.  Zinc Binding Modulates the Entire Folding Free Energy Surface of Human Cu,Zn Superoxide Dismutase 
Journal of molecular biology  2008;384(2):540-555.
Over 100 amino acid replacements in human Cu, Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially-folded states are primarily responsible for toxicity, the role of the structurally-important zinc ion in defining the folding free energy surface of dimeric SOD was determined by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with sub-nanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations towards more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (∼nanomolar). The significant decrease in the population of partially-folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a pre-organized zinc-binding loop in the transition state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.
PMCID: PMC2756654  PMID: 18840448
ALS; beta-barrel dimer; metal binding; protein folding; thermodynamics and kinetics
10.  Residual Structures, Conformational Fluctuations, and Electrostatic Interactions in the Synergistic Folding of Two Intrinsically Disordered Proteins 
PLoS Computational Biology  2012;8(1):e1002353.
To understand the interplay of residual structures and conformational fluctuations in the interaction of intrinsically disordered proteins (IDPs), we first combined implicit solvent and replica exchange sampling to calculate atomistic disordered ensembles of the nuclear co-activator binding domain (NCBD) of transcription coactivator CBP and the activation domain of the p160 steroid receptor coactivator ACTR. The calculated ensembles are in quantitative agreement with NMR-derived residue helicity and recapitulate the experimental observation that, while free ACTR largely lacks residual secondary structures, free NCBD is a molten globule with a helical content similar to that in the folded complex. Detailed conformational analysis reveals that free NCBD has an inherent ability to substantially sample all the helix configurations that have been previously observed either unbound or in complexes. Intriguingly, further high-temperature unbinding and unfolding simulations in implicit and explicit solvents emphasize the importance of conformational fluctuations in synergistic folding of NCBD with ACTR. A balance between preformed elements and conformational fluctuations appears necessary to allow NCBD to interact with different targets and fold into alternative conformations. Together with previous topology-based modeling and existing experimental data, the current simulations strongly support an “extended conformational selection” synergistic folding mechanism that involves a key intermediate state stabilized by interaction between the C-terminal helices of NCBD and ACTR. In addition, the atomistic simulations reveal the role of long-range as well as short-range electrostatic interactions in cooperating with readily fluctuating residual structures, which might enhance the encounter rate and promote efficient folding upon encounter for facile binding and folding interactions of IDPs. Thus, the current study not only provides a consistent mechanistic understanding of the NCBD/ACTR interaction, but also helps establish a multi-scale molecular modeling framework for understanding the structure, interaction, and regulation of IDPs in general.
Author Summary
Intrinsically disordered proteins (IDPs) are now widely recognized to play fundamental roles in biology and to be frequently associated with human diseases. Although the potential advantages of intrinsic disorder in cellular signaling and regulation have been widely discussed, the physical basis for these proposed phenomena remains sketchy at best. An integration of multi-scale molecular modeling and experimental characterization is necessary to uncover the molecular principles that govern the structure, interaction, and regulation of IDPs. In this work, we characterize the conformational properties of two IDPs involved in transcription regulation at the atomistic level and further examine the roles of these properties in their coupled binding and folding interactions. Our simulations suggest interplay among residual structures, conformational fluctuations, and electrostatic interactions that allows efficient synergistic folding of these two IDPs. In particular, we propose that electrostatic interactions might play an important role in facilitating rapid folding and binding recognition of IDPs, by enhancing the encounter rate and promoting efficient folding upon encounter.
PMCID: PMC3257294  PMID: 22253588
11.  Missense Mutation Lys18Asn in Dystrophin that Triggers X-Linked Dilated Cardiomyopathy Decreases Protein Stability, Increases Protein Unfolding, and Perturbs Protein Structure, but Does Not Affect Protein Function 
PLoS ONE  2014;9(10):e110439.
Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.
PMCID: PMC4207752  PMID: 25340340
12.  A Disorder-Induced Domino-Like Destabilization Mechanism Governs the Folding and Functional Dynamics of the Repeat Protein IκBα 
PLoS Computational Biology  2013;9(12):e1003403.
The stability of the repeat protein IκBα, a transcriptional inhibitor in mammalian cells, is critical in the functioning of the NF-κB signaling module implicated in an array of cellular processes, including cell growth, disease, immunity and apoptosis. Structurally, IκBα is complex, with both ordered and disordered regions, thus posing a challenge to the available computational protocols to model its conformational behavior. Here, we introduce a simple procedure to model disorder in systems that undergo binding-induced folding that involves modulation of the contact map guided by equilibrium experimental observables in combination with an Ising-like Wako-Saitô-Muñoz-Eaton model. This one-step procedure alone is able to reproduce a variety of experimental observables, including ensemble thermodynamics (scanning calorimetry, pre-transitions, m-values) and kinetics (roll-over in chevron plot, intermediates and their identity), and is consistent with hydrogen-deuterium exchange measurements. We further capture the intricate distance-dynamics between the domains as measured by single-molecule FRET by combining the model predictions with simple polymer physics arguments. Our results reveal a unique mechanism at work in IκBα folding, wherein disorder in one domain initiates a domino-like effect partially destabilizing neighboring domains, thus highlighting the effect of symmetry-breaking at the level of primary sequences. The offshoot is a multi-state and a dynamic conformational landscape that is populated by increasingly partially folded ensembles upon destabilization. Our results provide, in a straightforward fashion, a rationale to the promiscuous binding and short intracellular half-life of IκBα evolutionarily engineered into it through repeats with variable stabilities and expand the functional repertoire of disordered regions in proteins.
Author Summary
It is well recognized that unstructured or disordered proteins play a vital role in the cell. How does this disorder translate into function, and what effect does it have when linked to ordered regions? We attempt to answer this question by computationally characterizing the repeat protein IκBα, a central player in the NF-κB signaling module that possesses both structured and disordered domains. Upon constraining a structure-based statistical mechanical model with equilibrium experiments, we are able to successfully predict both the ensemble kinetic and single-molecule behaviors. Functionally, we unearth a unique mechanism in which the effect of disorder propagates, even into ordered regions, in a domino-like fashion, thus rendering the entire structure highly flexible. In other words, the evolutionarily designed disorder in IκBα places it on a functional precipice that can be either recruited for binding to transduce external stimuli or just be degraded to shut down the inhibitory effect, reconciling both functional and folding behaviors in a single framework.
PMCID: PMC3868533  PMID: 24367251
13.  Partitioning Conformational Intermediates Between Competing Refolding and Aggregation Pathways: Insights into Transthyretin Amyloid Disease† 
Biochemistry  2005;44(50):16612-16623.
Amyloid diseases are caused by the aberrant assembly of a protein in the extracellular space. Folded proteins are not amyloidogenic, however the native state is generally in equilibrium with a minor population of unfolded or partially folded aggregation-competent conformers outside of the cell. Understanding how the partially unfolded conformers kinetically partition between the competing refolding and aggregation pathways provides insight into how misfolding, which occurs continuously, becomes pathogenic. Towards this end we have previously studied the amyloidogenicity of transthyretin (TTR), a human β-sheet rich homotetrameric protein that must undergo rate-limiting tetramer dissociation and partial monomer unfolding to misassemble into amyloid and other aggregates. We demonstrate herein that TTR homotetramers reassemble by an unusual monomer-dimer-trimer-tetramer (MDRT) pathway. Therefore, the rate of every step in the reassembly pathway is dependent on the concentration of folded TTR monomer. Partitioning soluble TTR monomers between the reassembly pathway and the aggregation pathway should therefore depend on the relative concentrations of aggregates and assembly intermediates. Aggregate clearance is envisioned to play an important role in the partitioning of protein in vivo, where partitioning to the aggregation pathway becomes increasingly favorable under conditions where the concentration of aggregates is increased because aggregate clearance is slow relative to the rate of aggregation. This shift from efficient to inefficient aggregate clearance could occur with aging, offering an explanation for the age-associated nature of these neurodegenerative diseases.
PMCID: PMC2532856  PMID: 16342952
14.  Ligand Binding and Hydration in Protein Misfolding: Insights from Studies of Prion and p53 Tumor Suppressor Proteins† 
Accounts of Chemical Research  2009;43(2):271-279.
Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington’s disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues.
The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins.
Anfinsen’s paradigm that the structure of a protein is determined by its amino acid sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a disordered N-terminal domain and a highly flexible, not-well-packed C-terminal domain, which might account for its significant hydration. When PrP binds to biological molecules, such as glycosaminoglycans and nucleic acids, the disordered segments appear to fold and become less hydrated. Formation of the PrP−nucleic acid complex seems to accelerate the conversion of the cellular form of the protein into the disease-causing isoform. For p53, binding to some ligands, including nucleic acids, would prevent misfolding of the protein. Recently, several groups have begun to analyze the folding−misfolding of the individual domains of p53, but several questions remain unanswered. We discuss the implications of these findings for understanding the productive and incorrect folding pathways of these proteins in normal physiological states and in human disease, such as prion disorders and cancer. These studies are shown to lay the groundwork for the development of new drugs.
PMCID: PMC2825094  PMID: 19817406
15.  Kinetics and Thermodynamics of Membrane Protein Folding 
Biomolecules  2014;4(1):354-373.
Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.
PMCID: PMC4030980  PMID: 24970219
membrane proteins; thermodynamic stability; urea; guanidine hydrochloride; sodium dodecyl sulfate
16.  From the Test Tube to the Cell: Exploring the Folding and Aggregation of a β-Clam Protein 
Biopolymers  2007;88(2):157-163.
A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model β-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.
PMCID: PMC2904568  PMID: 17206628
protein folding; protein aggregation; fluorescence; in-cell folding; molecular crowding
17.  Implications from a Network-Based Topological Analysis of Ubiquitin Unfolding Simulations 
PLoS ONE  2008;3(5):e2149.
The architectural organization of protein structures has been the focus of intense research since it can hopefully lead to an understanding of how proteins fold. In earlier works we had attempted to identify the inherent structural organization in proteins through a study of protein topology. We obtained a modular partitioning of protein structures with the modules correlating well with experimental evidence of early folding units or “foldons”. Residues that connect different modules were shown to be those that were protected during the transition phase of folding.
Methodology/Principal Findings
In this work, we follow the topological path of ubiquitin through molecular dynamics unfolding simulations. We observed that the use of recurrence quantification analysis (RQA) could lead to the identification of the transition state during unfolding. Additionally, our earlier contention that the modules uncovered through our graph partitioning approach correlated well with early folding units was vindicated through our simulations. Moreover, residues identified from native structure as connector hubs and which had been shown to be those that were protected during the transition phase of folding were indeed more stable (less flexible) well beyond the transition state. Further analysis of the topological pathway suggests that the all pairs shortest path in a protein is minimized during folding.
We observed that treating a protein native structure as a network by having amino acid residues as nodes and the non-covalent interactions among them as links allows for the rationalization of many aspects of the folding process. The possibility to derive this information directly from 3D structure opens the way to the prediction of important residues in proteins, while the confirmation of the minimization of APSP for folding allows for the establishment of a potentially useful proxy for kinetic optimality in the validation of sequence-structure predictions.
PMCID: PMC2364640  PMID: 18478068
18.  Metal binding kinetics of bi-Histidine sites used in ψ-analysis: Evidence for high energy protein folding intermediates 
Biochemistry  2009;48(13):2950-2959.
The zinc-specific fluorophore, Zinpyr-1, is used in competition assays to determine the kinetic and thermodynamic parameters of Zn2+ binding to engineered bi-Histidine sites located in Ubiquitin and the B domain of protein A (BdpA). These binding sites are used in ψ-analysis studies to investigate structure formation in the folding transition state identified by the change in folding rate upon the addition of metal ions. For Ubiquitin, the on-rate binding constant and binding affinity for a site located along an α-helix are measured to be ~107 M-1s-1 and 3 μM, respectively. For a site located across two β-strands, the metal binding affinity was too weak to measure in the dye competition assays (Kd > 55 μM). The equilibrium-determined values for the Zn2+-induced stabilization of Ubiquitin and BdpA match the values derived from changes in the global folding and unfolding rates. Therefore, metal-ion binding is in fast equilibrium during the transit over the free energy barrier. Accordingly, the folding rate must be slower than the product of the fractional population of a high energy intermediate with the metal site formed and the metal binding on-rate constant. The known folding rate of 20 s-1 at 1.5 M guanidinium chloride in 400 μM Zn2+ provides an upper bound for the stability of such intermediates, ΔGU-I < +4 kcal·mol-1. These results support a view of the apparent two-state protein folding reaction surface as a fast pre-equilibrium between the denatured state and a series of high energy species. The net folding rate is a product of the equilibrium constant of the highest energy species and a transmission rate. For Ubiquitin, we estimate the transmission rate to be ~104 s-1. Implications to the role of unfolded chain diffusion on folding rates and barrier heights are discussed.
PMCID: PMC3313835  PMID: 19220047
Psi-analysis; Ubiquitin; free-energy barrier; kinetics
19.  Molecular Simulations of Cotranslational Protein Folding: Fragment Stabilities, Folding Cooperativity, and Trapping in the Ribosome 
PLoS Computational Biology  2006;2(7):e98.
Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.
The question of how proteins fold into their three-dimensional native conformations continues to be a subject of considerable interest, in large part because misfolding or aggregation of proteins is associated with a number of important diseases. Most previous research has focused on how proteins refold from denatured conformations in vitro, and much of the experimentally observed behavior has proven to be explicable with molecular simulations performed on computers. Recently attention has begun to move toward understanding protein folding as it occurs in vivo, which development requires, among other things, consideration of potential interactions with chaperonins and non-specific crowding effects due to the high macromolecular concentrations encountered in physiological conditions. Also under increasing consideration experimentally is the possibility that proteins might begin to fold while being synthesized (i.e., cotranslational folding), and the purpose of this work is therefore to develop and apply a first molecular simulation strategy capable of modeling this process. The simulations thus described, while not free of assumptions and approximations, nevertheless provide some intriguing glimpses into how the process of protein folding might be modulated through coupling to synthesis within the large ribosomal subunit.
PMCID: PMC1523309  PMID: 16789821
20.  In the Multi-domain Protein Adenylate Kinase, Domain Insertion Facilitates Cooperative Folding while Accommodating Function at Domain Interfaces 
PLoS Computational Biology  2014;10(11):e1003938.
Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.
Author Summary
Most individual protein domains fold in an all or nothing fashion. This cooperative folding is important because it reduces the existence of partially folded proteins which can stick to each other and create disease causing aggregates. However, numerous proteins have multiple domains, independent units of folding, stability and/or function. Several such proteins also fold cooperatively. It is thought that strong interactions between individual domains allow the folding to propagate from a nucleating domain to neighbouring ones and this enables cooperative folding in multi-domain proteins. Here, we computationally study the folding of the three-domain protein AKE and find instead that the topology of the protein, wherein the two less stable domains are inserted into the more stable one, promotes folding cooperativity. When the more stable domain is folded, the ends of the inserted domains are constrained and this allows them to fold easily. In such a protein topology, strong inter-domain interactions are not needed to promote folding cooperativity. Interface amino acids which would have been involved in ensuring that the domains fit together correctly can now be tuned for binding or catalysis or conformational transitions. Thus, inserted domains may be present in multi-domain proteins to promote both function and folding.
PMCID: PMC4230728  PMID: 25393408
21.  Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale 
PLoS Computational Biology  2013;9(12):e1003393.
After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded←→unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.
Author Summary
The delicate equilibrium between the folded and functional structure of a protein and its unfolded state is highly dependent on environmental variables such as the solvent. For example the co-solvent urea is a well-known protein denaturant that displaces the equilibrium towards unstructured and non-functional conformations of proteins. However the molecular mechanism behind its ability remains an enigma and the interpretation of the experimental data is still ambiguous. By analyzing a set of representative proteins through extensive molecular dynamics simulations in urea, we provide a robust and consensus picture of the first stages of urea-driven protein unfolding and elucidate the role of urea in accelerating protein unfolding. Our results suggest that urea, thanks to its stickiness and slow diffusion, benefits from the intrinsic flexibility of proteins and stabilizes partially open-states, slowly driving the protein toward unfolding.
PMCID: PMC3861036  PMID: 24348236
22.  BiP Clustering Facilitates Protein Folding in the Endoplasmic Reticulum 
PLoS Computational Biology  2014;10(7):e1003675.
The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as ‘clusters’). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.
Author Summary
The misfolding of proteins carries important implications for diseases such as Alzheimer's, Parkinson's, cancer, and diabetes. Once misfolded, proteins tend to associate into aggregates that pose a toxic threat to the cell. Chaperones are proteins that rescue the cell from an accumulation of these maladjusted proteins through dissociation of toxic oligomers and proper (re)folding. The endoplasmic reticulum (ER) is an organelle that serves as the staging ground for the chaperone activities of protein transport, folding, and maturation in the early secretory pathway. We have developed a computational model to investigate potential mechanisms that enable multiple ER-resident molecules working in concert to effectively fold peptides and transport nascent proteins across the ER membrane. Although previous models focused on chaperone interactions with peptides, we have explored the influence of cooperativity among chaperone molecules to assist in protein folding and maturation. We found that chaperone cooperation led to a higher yield of folded molecules compared to when chaperones bound to peptides in a 1∶1 stoichiometry. We have concluded that the clustering or multiple binding of chaperones may facilitate protein folding in vivo.
PMCID: PMC4081015  PMID: 24991821
23.  Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation 
eLife  2014;3:e02481.
The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system. Essential for disaggregation, the ClpB middle domain (MD) is a coiled-coil propeller that binds Hsp70. Although the ClpB subunit structure is known, positioning of the MD in the hexamer and its mechanism of action are unclear. We obtained electron microscopy (EM) structures of the BAP variant of ClpB that binds the protease ClpP, clearly revealing MD density on the surface of the ClpB ring. Mutant analysis and asymmetric reconstructions show that MDs adopt diverse positions in a single ClpB hexamer. Adjacent, horizontally oriented MDs form head-to-tail contacts and repress ClpB activity by preventing Hsp70 interaction. Tilting of the MD breaks this contact, allowing Hsp70 binding, and releasing the contact in adjacent subunits. Our data suggest a wavelike activation of ClpB subunits around the ring.
eLife digest
Proteins are long chain-like molecules that twist and fold into complex three-dimensional shapes in order to carry out their functions. High temperatures or other types of stress can cause proteins to fold incorrectly, and misfolded proteins can form clumps (or aggregates) that are harmful to cells. Additional proteins called chaperones are therefore used by cells to help proteins to fold correctly, or to refold poorly folded proteins.
ClpB proteins (and related proteins) are chaperones found in bacteria, fungi and plants; these proteins co-operate with other chaperones to rescue misfolded proteins that have aggregated—an activity that helps cells to survive heat shock and other stresses. Six ClpB proteins work together to form a ring-shaped complex, and the misfolded protein is unfolded by threading it through the centre of this ring. Each ClpB protein also has a middle domain that acts to switch the complex on and off as needed.
The middle domains are known to form coiled-coils, with protein helices coiled together like the strands of a rope. However, previous efforts to work out the structure of the ClpB complex did not clearly establish where these coiled-coils were positioned relative to the rest of the ring.
Now Carroni et al. have used image processing to overcome these problems and reveal that the middle domains are wrapped around the outer edge of the ring complex. Analysis of ClpB mutants that lock the complex in either an off or on state revealed that the middle domains are linked head-to tail to encircle the ring when the complex is off. However, when the complex switches on, the middle domains let go of each other and tilt, allowing the ring to change shape. Carroni et al. suggest that the exposed ends of the middle domains are free to bind to other chaperones (those that work to refold the unfolded proteins), thereby activating the complex.
Although Carroni et al. have revealed how the ClpB ring complex is activated, further work is needed to understand exactly how the unlocked ring works to rescue misfolded proteins from aggregates within cells.
PMCID: PMC4023160  PMID: 24843029
single particle EM; ClpB/Hsp104; protein unfolding; E. coli; S. cerevisiae
24.  Mechanically Untying a Protein Slipknot: Multiple Pathways Revealed by Force Spectroscopy and Steered Molecular Dynamics Simulations 
Journal of the American Chemical Society  2012;134(25):10428-10435.
Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechancial unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie, and the polypeptide chain to completely extend. These distinct unfolding pathways proceed either via a two-state or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus provding a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechancial unfolding pathway of AFV3-109, and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding.
PMCID: PMC3663486  PMID: 22626004
25.  Structure-Based, Rational Design of T Cell Receptors 
Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157–165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, KD = ∼1 − 5 μM. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function, in line with the “half-life” model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.
PMCID: PMC3770923  PMID: 24062738
molecular modeling; protein-engineering; TCR; TCR-pMHC; immunotherapy; adoptive transfer; cancer

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