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1.  Energy Functions in De Novo Protein Design: Current Challenges and Future Prospects 
Annual review of biophysics  2013;42:10.1146/annurev-biophys-083012-130315.
In the past decade, a concerted effort to successfully capture specific tertiary packing interactions produced specific three-dimensional structures for many de novo designed proteins that are validated by nuclear magnetic resonance and/or X-ray crystallographic techniques. However, the success rate of computational design remains low. In this review, we provide an overview of experimentally validated, de novo designed proteins and compare four available programs, RosettaDesign, EGAD, Liang-Grishin, and RosettaDesign-SR, by assessing designed sequences computationally. Computational assessment includes the recovery of native sequences, the calculation of sizes of hydrophobic patches and total solvent-accessible surface area, and the prediction of structural properties such as intrinsic disorder, secondary structures, and three-dimensional structures. This computational assessment, together with a recent community-wide experiment in assessing scoring functions for interface design, suggests that the next-generation protein-design scoring function will come from the right balance of complementary interaction terms. Such balance may be found when more negative experimental data become available as part of a training set.
PMCID: PMC3851009  PMID: 23451890
computational assessment of designed sequences; secondary structure; solvent accessibility; hydrophobic patch; intrinsic disorder
2.  Structural and Energetic Basis of Allostery 
Annual review of biophysics  2012;41:585-609.
Allostery is a biological phenomenon of fundamental importance in regulation and signaling, and efforts to understand this process have led to the development of numerous models. In spite of individual successes in understanding the structural determinants of allostery in well-documented systems, much less success has been achieved in identifying a set of quantitative and transferable ground rules that provide an understanding of how allostery works. Are there organizing principles that allow us to relate structurally different proteins, or are the determinants of allostery unique to each system? Using an ensemble-based model, we show that allosteric phenomena can be formulated in terms of conformational free energies of the cooperative elements in a protein and the coupling interactions between them. Interestingly, the resulting allosteric ground rules provide a framework to reconcile observations that challenge purely structural models of site-to-site coupling, including (a) allostery in the absence of pathways of structural distortions, (b) allostery in the absence of any structural change, and (c) the ability of allosteric ligands to act as agonists under some circumstances and antagonists under others. The ensemble view of allostery that emerges provides insights into the energetic prerequisites of site-to-site coupling and thus into how allostery works.
PMCID: PMC3935618  PMID: 22577828
protein thermodynamics; conformational change; protein function; signal transduction; agonism; antagonism
3.  Quantitative modeling of bacterial chemotaxis: Signal amplification and accurate adaptation 
Annual review of biophysics  2013;42:337-359.
We review the recent developments in understanding bacterial chemotaxis signaling pathway by using quantitative modeling methods. The models are developed based on structural information of the signaling complex and the dynamics of the underlying biochemical network. We focus on two important functions of the bacterial chemotaxis pathway: signal amplification and adaptation. We give in-depth description of the structure and the dynamics of the mathematical models and their comparison with existing experiments, with emphasis on the predictability of the models. Finally, we point out several directions for future development of the modeling approach in understanding the bacterial chemo-sensory system.
PMCID: PMC3737589  PMID: 23451887
Bacterial Chemotaxis; Gradient Sensing; Receptor Clustering; Adaptation; Modeling
4.  Influences of Membrane Mimetic Environments on Membrane Protein Structures 
Annual review of biophysics  2013;42:361-392.
The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins.
PMCID: PMC3731949  PMID: 23451886
structure determination; membrane biophysical properties; structural perturbations; bilayer and detergent environments; native structures; structural enhancement
5.  Single Molecule Views of Protein Movement on Single Stranded DNA 
Annual review of biophysics  2012;41:295-319.
The advent of new technologies allowing the study of single biological molecules continues to have a major impact on studies of interacting systems as well as enzyme reactions. These approaches (fluorescence, optical and magnetic “tweezers”), in combination with ensemble methods, have been particularly useful for mechanistic studies of protein-nucleic acid interactions and enzymes that function on nucleic acids. We review progress in the use of single molecule methods to observe and perturb the activities of proteins and enzymes that function on flexible single stranded DNA. These include single stranded (ss)DNA binding (SSB) proteins, recombinases (RecA/Rad51) and helicases/translocases that operate as motor proteins and play central roles in genome maintenance. We emphasize methods that have been used to detect and study the movement of these proteins (both ATP-dependent directional and random movement) along the ssDNA and the mechanistic and functional information that can result from detailed analysis of such movement.
PMCID: PMC3719979  PMID: 22404684
FRET; helicases; RecA; Rad51; SSB; optical tweezers
6.  Doing Molecular Biophysics: Finding, Naming, and Picturing Signal Within Complexity 
A macromolecular structure, as measured data or as a list of coordinates or even on-screen as a full atomic model, is an extremely complex and confusing object. The underlying rules of how it folds, moves, and interacts as a biological entity are even less evident or intuitive to the human mind. To do science on such molecules, or to relate them usefully to higher levels of biology, we need to start with a natural history that names their features in meaningful ways and with multiple representations (visual or algebraic) that show some aspect of their organizing principles. The two of us have jointly enjoyed a highly varied and engrossing career in biophysical research over nearly 50 years. Our frequent changes of emphasis are tied together by two threads: first, by finding the right names, visualizations, and methods to help both ourselves and others to better understand the 3D structures of protein and RNA molecules, and second, by redefining the boundary between signal and noise for complex data, in both directions—sometimes identifying and promoting real signal up out of what seemed just noise, and sometimes demoting apparent signal into noise or systematic error. Here we relate parts of our scientific and personal lives, including ups and downs, influences, anecdotes, and guiding principles such as the title theme.
PMCID: PMC3695750  PMID: 23451888
scientific biography; structural biology; molecular graphics; ribbon drawings; structure validation; all-atom contacts
7.  Phase separation in biological membranes: integration of theory and experiment 
Annual review of biophysics  2010;39:207-226.
Lipid bilayer model membranes can undergo transitions between ordered and disordered phases, and membranes that contain a mixture of lipid species can undergo phase separations. Studies of these transformations are of interest for what they can tell us about the interaction energies of lipid molecules of different species and conformations. Nanoscopic phases can provide a model for membrane rafts, which have important biological functions in cell membranes. Important questions are whether lipid nanodomains can exist in stable equilibrium in membranes and what is the distribution of their sizes in membranes of different composition. It is also important to know the lifetimes of nanodomains. Theoretical methods have supplied much important information on these questions, but better experimental methods are needed to detect and characterize nanodomains under normal membrane conditions. This review summarizes linkages between theoretical and experimental studies of phase separation in lipid bilayer model membranes.
PMCID: PMC3694198  PMID: 20192775
8.  Molecular Basis of NF-κB Signaling 
Annual review of biophysics  2013;42:443-468.
NF-κB (nuclear factor kappa B) family transcription factors are master regulators of immune and inflammatory processes in response to both injury and infection. In the latent state, NF-κBs are sequestered in the cytosol by their inhibitor IκB (inhibitor of NF-κB) proteins. Upon stimulations of innate immune receptors such as Toll-like receptors and cytokine receptors such as those in the TNF (tumor necrosis factor) receptor superfamily, a series of membrane proximal events lead to the activation of the IKK (IκB kinase). Phosphorylation of IκBs results in their proteasomal degradation and the release of NF-κB for nuclear translocation and activation of gene transcription. Here, we review the plethora of structural studies in these NF-κB activation pathways, including the TRAF (TNF receptor–associated factor) proteins, IKK, NF-κB, ubiquitin ligases, and deubiquitinating enzymes. Although these structures only provide snapshots of isolated processes, an emerging picture is that these signaling cascades coalesce into large oligomeric signaling complexes, or signalosomes, for signal propagation.
PMCID: PMC3678348  PMID: 23495970
TNF; Toll-like receptor; IKK; TRAF; ubiquitin; NEMO
9.  Virus Maturation 
Annual review of biophysics  2012;41:473-496.
We examined virus maturation of selected non-enveloped and enveloped ssRNA viruses; retroviruses; bacteriophages and herpes virus. Processes associated with maturation in the RNA viruses range from subtle (noda and picornaviruses) to dramatic (tetraviruses and togaviruses). The elaborate assembly and maturation pathway of HIV is discussed in contrast to the less sophisticated but highly efficient processes associated with togaviruses. Bacteriophage assembly and maturation are discussed in general terms with specific examples chosen for emphasis. Finally the herpes viruses are compared with bacteriophages. The data support divergent evolution of noda, picorna and tetraviruses from a common ancestor and divergent evolution of alpha and flaviviruses from a common ancestor. Likewise, bacteriophages and herpes viruses almost certainly share a common ancestor in their evolution. Comparing all the viruses, we conclude that maturation is a convergent process that is required to solve conflicting requirements in biological dynamics and function.
PMCID: PMC3607295  PMID: 22404678
10.  Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase 
Annual review of biophysics  2011;41:81-101.
Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies.
PMCID: PMC3575517  PMID: 22224599
Diacylglycerol kinase; Undecaprenol kinase; dgkA gene; Membrane protein structure; Membrane protein folding
11.  Bioimage informatics for experimental biology 
Annual review of biophysics  2009;38:327-346.
Over the last twenty years there have been great advances in light microscopy with the result that multi-dimensional imaging has driven a revolution in modern biology. The development of new approaches of data acquisition are reportedly frequently, and yet the significant data management and analysis challenges presented by these new complex datasets remains largely unsolved. Like the well-developed field of genome bioinformatics, central repositories are and will be key resources, but there is a critical need for informatics tools in individual laboratories to help manage, share, visualize, and analyze image data. In this article we present the recent efforts by the bioimage informatics community to tackle these challenges and discuss our own vision for future development of bioimage informatics solution.
PMCID: PMC3522875  PMID: 19416072
microscopy; file formats; image management; image analysis; image processing
12.  Bacterial Mechanosensitive Channels—MscS: Evolution’s Solution to Creating Sensitivity in Function 
Annual review of biophysics  2012;41:157-177.
The discovery of mechanosensing channels has changed our understanding of bacterial physiology. The mechanosensitive channel of small conductance (MscS) is perhaps the most intensively studied of these channels. MscS has at least two states: closed, which does not allow solutes to exit the cytoplasm, and open, which allows rapid efflux of solvent and solutes. The ability to appropriately open or close the channel (gating) is critical to bacterial survival. We briefly review the science that led to the isolation and identification of MscS. We concentrate on the structure-function relationship of the channel, in particular the structural and biochemical approaches to understanding channel gating. We highlight the troubling discrepancies between the various models developed to understand MscS gating.
PMCID: PMC3378650  PMID: 22404681
protein structure; modeling; crystallography; EPR; mutagenesis; ion channel
13.  Lessons Learned From UvrD Helicase : Mechanism For Directional Movement 
Annual review of biophysics  2010;39:367-385.
How do molecular motors convert chemical energy to mechanical work? Helicases and nucleic acids offer simple motor systems for extensive biochemical and biophysical analyses. Atomic resolution structures of UvrD-like helicases complexed with DNA in the presence of AMPPNP, ADP·Pi, and Pi reveal several salient points that aid understanding mechano-chemical coupling. Each ATPase cycle causes two motor-domains to rotationally close and open. At a minimum, two motor-track contact points of alternating tight and loose attachment convert domain rotations to uni-directional movement. A motor is “in gear” for action only when fully in contact with its track and, if applicable, working against a load. The orientation of domain rotation relative to the track determines whether the movement is linear, spiral or circular. Motors powered by ATPases likely deliver each power stroke in two parts, before and after ATP hydrolysis. Implications of these findings for analyzing hexameric helicase, F1F0 ATPase and kinesin are discussed.
PMCID: PMC3480338  PMID: 20192763
motor-track-load; mechano-chemical coupling; ATPase; linear; spiral; circular; step size; inchworm; hand-over-hand
14.  The Cyanobacterial Circadian System: From Biophysics to Bioevolution 
Annual review of biophysics  2011;40:143-167.
Recent studies have unveiled the molecular machinery responsible for the biological clock in cyanobacteria and found that it exerts pervasive control over cellular processes including global gene expression. Indeed, the entire chromosome undergoes daily cycles of topology/compaction! The circadian system comprises both a posttranslational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO can be reconstituted in vitro with three purified proteins (KaiA, KaiB, and KaiC) and ATP. These are the only circadian proteins for which high-resolution structures are available. Phase in this nanoclockwork has been associated with key phosphorylations of KaiC. Structural considerations illuminate the mechanism by which the KaiABC oscillator ratchets unidirectionally. Models of the complete in vivo system have important implications for our understanding of circadian clocks in higher organisms, including mammals. The conjunction of structural, biophysical, and biochemical approaches to this system has brought our understanding of the molecular mechanisms of biological timekeeping to an unprecedented level.
PMCID: PMC3093959  PMID: 21332358
cyanobacteria; Kai; KaiABC; cell division; in vitro oscillators
15.  Molecular Mechanisms of Ubiquitin-Dependent Membrane Traffic 
Annual Review of Biophysics  2011;40:119-142.
Over the past fourteen years, ubiquitination has emerged as a centrally important mechanism governing the subcellular trafficking of proteins. Ubiquitination, interaction with sorting factors that contain ubiquitin binding domains, and finally deubiquitination govern the itineraries of cargo proteins that include yeast carboxypeptidase S, the epithelial sodium channel ENaC, and epidermal growth factor receptor. The molecular structures and mechanisms of the paradigmatic HECT and RING domain ubiquitin ligases, JAMM and USP domain deubiquitinating enzymes, and numerous ubiquitin binding domains involved in these pathways, have been worked out in recent years and are described.
PMCID: PMC3272705  PMID: 21332354
Lysosome; vacuole; yeast genetics; EGF; EGF receptor; growth factor receptor; epithelial sodium channel; ENaC; yeast genetics; carboxypeptidase S; protein structure; crystal structure; ubiquitin; RING domain; HECT domain; JAMM domain; isopeptidase; ubiquitin ligase; deubiquitinating enzyme; ubiquitin binding domain; ESCRT complex
16.  Lipids and Cholesterol as Regulators of Traffic in the Endomembrane System 
Annual Review of Biophysics  2010;39:559-578.
The endomembrane system of eukaryotic cells uses membrane-enclosed carriers to move diverse macromolecules among different membrane-bound compartments, a requirement for cells to secrete and take up molecules from their environment. Two recycling pathways—biosynthetic and endocytic, each with specific lipid components—make up this system, with the Golgi apparatus mediating transport between the two. Here, we integrate lipid-based mechanisms into the description of this system. A partitioning model of the Golgi apparatus is discussed as a working hypothesis to explain how membrane lipids and proteins that are segregated based on lateral lipid partitioning support the unique composition of the biosynthetic and endocytic recycling pathways in the face of constant trafficking of molecular constituents. We further discuss how computational modeling can allow for interpretation of experimental findings and provide mechanistic insight into these important cellular pathways.
PMCID: PMC3366628  PMID: 20192772
sphingolipids; microdomains; Golgi; secretion; endocytosis partitioning; modeling
17.  Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution 
Annual Review of Biophysics  2010;39:185-205.
Some bacteria contain organelles or microcompartments consisting of a large virion-like protein shell encapsulating sequentially acting enzymes. These organized microcompartments serve to enhance or protect key metabolic pathways inside the cell. The variety of bacterial microcompartments provide diverse metabolic functions, ranging from CO2 fixation to the degradation of small organic molecules. Yet they share an evolutionarily related shell, which is defined by a conserved protein domain that is widely distributed across the bacterial kingdom. Structural studies on a number of these bacterial microcompartment shell proteins are illuminating the architecture of the shell and highlighting its critical role in controlling molecular transport into and out of microcompartments. Current structural, evolutionary, and mechanistic ideas are discussed, along with genomic studies for exploring the function and diversity of this family of bacterial organelles.
PMCID: PMC3272493  PMID: 20192762
carboxysome; molecular transport; protein assembly; nanocompartments; protein shell
18.  Imaging Transcription in Living Cells 
Annual review of biophysics  2009;38:173-196.
The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.
PMCID: PMC3166783  PMID: 19416065
19.  Actin Structure and Function 
Annual review of biophysics  2011;40:169-186.
Actin is the most abundant protein in most eukaryotic cells. It is highly conserved and participates in more protein-protein interactions than any known protein. These properties, along with its ability to transition between monomeric (G-actin) and filamentous (F-actin) states under the control of nucleotide hydrolysis, ions, and a large number of actin-binding proteins, make actin a critical player in many cellular functions, ranging from cell motility and the maintenance of cell shape and polarity to the regulation of transcription. Moreover, the interaction of filamentous actin with myosin forms the basis of muscle contraction. Owing to its central role in the cell, the actin cytoskeleton is also disrupted or taken over by numerous pathogens. Here we review structures of G- and F-actin and discuss some of the interactions that control the polymerization and disassembly of actin.
PMCID: PMC3130349  PMID: 21314430
X-ray crystallography; electron microscopy; fiber diffraction; actin-binding-proteins
20.  Single-Molecule Approaches to Stochastic Gene Expression 
Annual review of biophysics  2009;38:255-270.
Both the transcription of mRNAs from genes and their subsequent translation into proteins are inherently stochastic biochemical events, and this randomness can lead to substantial cell-to-cell variability in mRNA and protein numbers in otherwise identical cells. Recently, a number of studies have greatly enhanced our understanding of stochastic processes in gene expression by utilizing new methods capable of counting individual mRNAs and proteins in cells. In this review, we examine the insights that these studies have yielded in the field of stochastic gene expression. In particular, we discuss how these studies have played in understanding the properties of bursts in gene expression. We also compare the array of different methods that have arisen for single mRNA and protein detection, highlighting their relative strengths and weaknesses. In conclusion, we point out further areas where single-molecule techniques applied to gene expression may lead to new discoveries.
PMCID: PMC3126657  PMID: 19416069
molecular counting; single cell; single mRNA; single protein; random; mRNA; protein; cell-to-cell variability; gene expression noise; noisy gene expression
21.  Chromatin Dynamics 
Annual review of biophysics  2010;39:471-489.
The expression patterns of many protein-coding genes are orchestrated in response to exogenous stimuli, as well as cell-type-specific developmental programs. In recent years, researchers have shown that dynamic chromatin movements and interactions in the nucleus play a crucial role in gene regulation. In this review, we highlight our current understanding of the organization of chromatin in the interphase nucleus and the impact of chromatin dynamics on gene expression. We also discuss the current state of knowledge with regard to the localization of active and inactive genes within the three-dimensional nuclear space. Furthermore, we address recent findings that demonstrate the movements of chromosomal regions and genomic loci in association with changes in transcriptional activity. Finally, we discuss the role of intra-and interchromosomal interactions in the control of coregulated genes.
PMCID: PMC2894465  PMID: 20462379
nuclear organization; gene positioning; gene expression; chromosome territory
22.  Actin Dynamics: From Nanoscale to Microscale 
Annual review of biophysics  2010;39:91-110.
The dynamic nature of actin in cells manifests itself in many ways: Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The rate of actin incorporation at the leading edge of a moving cell is roughly consistent with existing theories, and the factors determining the characteristic time of actin polymerization are fairly well understood. However, our understanding of actin disassembly is limited, in particular the interplay between severing and depolymerization and the role of specific combinations of proteins in implementing disassembly events. The origins of cell-edge oscillations, and their possible relation to actin waves, are a fruitful area of future research.
PMCID: PMC2967719  PMID: 20462375
polymerization; branched network; severing; overshoot; actin turnover; reaction-diffusion waves
23.  Rewiring Cells: Synthetic biology as a tool to interrogate the organizational principles of living systems 
Annual review of biophysics  2010;39:515-537.
The living cell is an incredibly complex entity, and the goal of predictively and quantitatively understanding its function is one of the next great challenges in biology. Much of what we know about the cell concerns its constituent parts, but to a great extent, we have yet to decode how these parts are organized to yield complex physiological function. Classically, we have learned about the organization of cellular networks by perturbing them through genetic or chemical means. The emerging discipline of synthetic biology offers an additional, powerful way to study systems. By rearranging the parts that comprise existing networks, we can gain valuable insight into the hierarchical logic of the networks and identify the modular building blocks that evolution uses to generate innovative function. Additionally, by building minimal “toy” networks, one can systematically explore the relationship between network space (linkages and parameters) and functional space (the system's physiological behavior). Here, we outline recent work that uses synthetic biology approaches to investigate the organization and function of cellular networks, and describe a vision for a synthetic biology toolkit that could be used to interrogate the design principles of diverse systems.
PMCID: PMC2965450  PMID: 20192780
24.  Global Dynamics of Proteins: Bridging Between Structure and Function 
Biomolecular systems possess unique, structure-encoded dynamic properties that underlie their biological functions. Recent studies indicate that these dynamic properties are determined to a large extent by the topology of native contacts. In recent years, elastic network models used in conjunction with normal mode analyses have proven to be useful for elucidating the collective dynamics intrinsically accessible under native state conditions, including in particular the global modes of motions that are robustly defined by the overall architecture. With increasing availability of structural data for well-studied proteins in different forms (liganded, complexed, or free), there is increasing evidence in support of the correspondence between functional changes in structures observed in experiments and the global motions predicted by these coarse-grained analyses. These observed correlations suggest that computational methods may be advantageously employed for assessing functional changes in structure and allosteric mechanisms intrinsically favored by the native fold.
PMCID: PMC2938190  PMID: 20192781
elastic network models; normal modes; principal component analysis; collective motions; allosteric changes in conformation; closed/open conformations
25.  Site-Directed Spectroscopic Probes of Actomyosin Structural Dynamics 
Annual review of biophysics  2009;38:347-369.
Spectroscopy of myosin and actin has entered a golden age. High-resolution crystal structures of isolated actin and myosin have been used to construct detailed models for the dynamic actomyosin interactions that move muscle. Improved protein mutagenesis and expression technologies have facilitated site-directed labeling with fluorescent and spin probes. Spectroscopic instrumentation has achieved impressive advances in sensitivity and resolution. Here we highlight the contributions of site-directed spectroscopic probes to understanding the structural dynamics of myosin II and its actin complexes in solution and muscle fibers. We emphasize studies that probe directly the movements of structural elements within the myosin catalytic and light-chain domains, and changes in the dynamics of both actin and myosin due to their alternating strong and weak interactions in the ATPase cycle. A moving picture emerges in which single biochemical states produce multiple structural states, and transitions between states of order and dynamic disorder power the actomyosin engine.
PMCID: PMC2928672  PMID: 19416073
EPR; spin label; fluorescence; luminescence; disorder; mobility

Results 1-25 (32)