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1.  Effects of spermine NONOate and ATP on protein aggregation: light scattering evidences 
BMC Biophysics  2013;6:1.
Background and objective
Regulating protein function in the cell by small molecules, provide a rapid, reversible and tunable tool of metabolic control. However, due to its complexity the issue is poorly studied so far. The effects of small solutes on protein behavior can be studied by examining changes of protein secondary structure, in its hydrodynamic radius as well as its thermal aggregation. The study aim was to investigate effects of adenosine-5’-triphosphate (ATP), spermine NONOate (NO donor) as well as sodium/potassium ions on thermal aggregation of albumin and hemoglobin. To follow aggregation of the proteins, their diffusion coefficients were measured by quasi-elastic light scattering (QELS) at constant pH (7.4) in the presence of solutes over a temperature range from 25°C to 80°C.
Results and discussion
1) Spermine NONOate persistently decreased the hemoglobin aggregation temperature Tairrespectively of the Na+/K+ environment, 2) ATP alone had no effect on the protein’s thermal stability but it facilitated protein’s destabilization in the presence of spermine NONOate and 3) mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions.
The ATP effect on protein aggregation was ambiguous: ATP alone had no effect on the protein’s thermal stability but it facilitated protein’s destabilization in the presence of nitric oxide. The magnitude and direction of the observed effects strongly depended on concentrations of K+ and Na+ in the solution.
PMCID: PMC3561150  PMID: 23289636
2.  Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7 
AMB Express  2014;4:42.
Biofilms in the industrial environment could be problematic. Encased in extracellular polymeric substances, pathogens within biofilms are significantly more resistant to chlorine and other disinfectants. Recent studies suggest that compounds capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of sessile bacteria and provide a foundation for novel approaches to controlling biofilms formed by some microorganisms. In this work, we compared the ability of five nitric oxide donors (molsidomine, MAHMA NONOate, diethylamine NONOate, diethylamine NONOate diethylammonium salt, spermine NONOate) to dislodge biofilms formed by non-typhoidal Salmonella enterica and pathogenic E. coli on plastic and stainless steel surfaces at different temperatures. All five nitric oxide donors induced significant (35-80%) dispersal of biofilms, however, the degree of dispersal and the optimal dispersal conditions varied. MAHMA NONOate and molsidomine were strong dispersants of the Salmonella biofilms formed on polystyrene. Importantly, molsidomine induced dispersal of up to 50% of the pre-formed Salmonella biofilm at 4°C, suggesting that it could be effective even under refrigerated conditions. Biofilms formed by E. coli O157:H7 were also significantly dispersed. Nitric oxide donor molecules were highly active within 6 hours of application. To better understand mode of action of these compounds, we identified Salmonella genomic region recA-hydN, deletion of which led to an insensitivity to the nitric oxide donors.
PMCID: PMC4070026  PMID: 24995149
Biofilm control; Bacterial signaling; Food-borne pathogens; Nitric oxide
3.  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
4.  Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)α1-specific activity and glucose transport in human skeletal muscle 
Diabetologia  2010;53(6):1142-1150.
We investigated the direct effect of a nitric oxide donor (spermine NONOate) on glucose transport in isolated human skeletal muscle and L6 skeletal muscle cells. We hypothesised that pharmacological treatment of human skeletal muscle with N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport.
Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 µmol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction was determined.
In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p < 0.05), concomitant with increased cGMP levels (80-fold, p < 0.001). Phosphorylation of components of the canonical insulin signalling cascade was unaltered by spermine NONOate exposure, implicating an insulin-independent signalling mechanism. Consistent with this, spermine NONOate increased AMP-activated protein kinase (AMPK)-α1-associated activity (1.7-fold, p < 0.05). In L6 muscle cells, spermine NONOate increased glucose uptake (p < 0.01) and glycogen synthesis (p < 0.001), an effect that was in addition to that of insulin. Spermine NONOate also elicited a concomitant increase in AMPK and acetyl-CoA carboxylase phosphorylation. In the presence of the guanylate cyclase inhibitor LY-83583 (10 µmol/l), spermine NONOate had no effect on glycogen synthesis and AMPK-α1 phosphorylation.
Pharmacological treatment of skeletal muscle with spermine NONOate increases glucose transport via insulin-independent signalling pathways involving increased intracellular cGMP levels and AMPK-α1-associated activity.
PMCID: PMC2860569  PMID: 20349036
Contraction; Exercise; GLUT4; Spermine NONOate
5.  Buffer-dependent fragmentation of a humanized full-length monoclonal antibody 
Journal of pharmaceutical sciences  2010;99(7):2962-2974.
During storage stability studies of a monoclonal antibody (mAb) it was determined that the primary route of degradation involved fragmentation into lower molecular weight species. The fragmentation was characterized with size-exclusion high performance liquid chromatography (SE-HPLC), SDS-PAGE and matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Fragmentation proceeded via hydrolysis, likely catalyzed by trace metal ions, of a peptide bond in the hinge region of the mAb’s heavy chain, which produced two prominent low molecular weight species during storage: a single, free Fab fragment and a Fab+Fc fragment. The fragmentation is observed in phosphate-buffered solutions at two ionic strengths but not in histidine-buffered solutions at identical ionic strengths. Chaotrope-induced and thermally-induced unfolding studies of the mAb indicated differences in the unfolding pathways between the two buffer solutions. This folding intermediate observed during chaotrope-induced unfolding was further characterized by intrinsic fluorescence quenching, which suggested that a small portion of the molecule is resistant to chaotrope-induced unfolding in histidine buffer systems. The thermally-induced unfolding indicates a reduction in cooperativity of the unfolding process in the presence of histidine relative to phosphate. A relationship between the histidine-induced effects on unfolding pathway and the relative resistance to fragmentation is suggested.
PMCID: PMC3938388  PMID: 20091831
6.  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
7.  Thermal, Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation 
PLoS ONE  2014;9(8):e103579.
Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol−1 and 14.90 Kcal mol−1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp) is 3.42 Kcal mol−1 K−1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.
PMCID: PMC4139268  PMID: 25140525
8.  Biophysical Analysis of Progressive C-Terminal Truncations of Human Apolipoprotein E4: Insights into Secondary Structure and Unfolding Properties 
Biochemistry  2008;47(35):9071-9080.
Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer’s disease and has been associated with a variety of neuropathological processes. ApoE4 C-terminally truncated forms have been found in brains of Alzheimer’s disease patients. Structural rearrangements in apoE4 are known to be key to its physiological functions. To understand the effect of C-terminal truncations on apoE4 lipid-free structure, we produced a series of recombinant apoE4 forms with progressive C-terminal deletions between residues 166 and 299. Circular dichroism measurements show a dramatic loss in helicity upon removal of the last 40 C-terminal residues, whereas further truncations of residues 203–259 lead to recovery of helical content. Further deletion of residues 186–202 leads to a small increase in helical content. Thermal denaturation indicated that removal of residues 260–299 leads to an increase in melting temperature but truncations down to residue 186 did not further affect the melting temperature. The progressive C-terminal truncations, however, gradually increased the cooperativity of thermal unfolding. Chemical denaturation of the apoE4 forms revealed a two-step process with a clear intermediate stage that is progressively lost as the C-terminus is truncated down to residue 230. Hydrophobic fluorescent probe binding suggested that regions 260–299 and 186–202 contain hydrophobic sites, the former being solvent accessible in the wild-type molecule and the latter being accessible only upon truncation. Taken together, our results show an important but complex role of apoE4 C-terminal segments in secondary structure stability and unfolding and suggest that interactions mediated by the C-terminal segments are important for the structural integrity and conformational changes of apoE4.
PMCID: PMC2692411  PMID: 18690708
9.  A Biochemical-Biophysical Study of Hemoglobins from Woolly Mammoth, Asian Elephant, and Humans† 
Biochemistry  2011;50(34):7350-7360.
This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene Ice-ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A2 (a minor component of human Hb). We have obtained oxygen equilibrium curves and calculated O2 affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, 1H-NMR spectroscopy data indicates that both α1(β/δ)1 and α1(β/δ)2 interfaces in rHb WM and rHb AE are perturbed, whereas only the α1δ1 interface in Hb A2 is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.
PMCID: PMC3160526  PMID: 21806075
10.  Hydroxyurea stimulates the release of ATP from rabbit erythrocytes through an increase in calcium and nitric oxide production 
European journal of pharmacology  2010;645(0):32-38.
Hydroxyurea, a proven therapy for sickle cell disease, is known to improve blood flow and reduce vaso-occlusive crises, although its exact mechanism of action is not clear. The objective of this study was to determine if hydroxyurea results in an increase of ATP release from the red blood cell (RBC) via the drug's ability to stimulate nitric oxide (NO) production in these cells. A system enabling the flow of RBCs through microbore tubing was used to investigate ATP release from the RBC. Incubation of rabbit RBCs (7% hct) with 50 μM hydroxyurea resulted in a significant increase in the release of ATP from these cells. This level of ATP release was not detected in the absence of flow. Studies also showed that increments in hydroxyurea and NO (from spermineNONOate) resulted in an initial increase in ATP release, followed by a decrease in this release at higher concentrations of hydroxyurea and the NO donor. Incubation with L-NAME abolished the effect of the hydroxyurea, suggesting that NO production by the RBC was involved. Indeed, in the presence of 50 μM hydroxyurea, the amount of total Ca2+ measured (by atomic absorption spectroscopy) in a 7% solution of RBCs increased from 363 ± 47 ng/ml and 530 ± 52 ng/ml. Finally, EPR studies suggest that an increase in nitrosylated Hb in the RBC is only measured for those studies involving hydroxyurea and a Ca2+-containing buffer.
PMCID: PMC4051288  PMID: 20655902
nitric oxide; hydroxyurea; ATP; eNOS; red cells; sickle cell
11.  Measurement of the Effect of Monovalent Cations on RNA Hairpin Stability 
Journal of the American Chemical Society  2007;129(48):14966-14973.
Using optical tweezers, we have measured the effect of monovalent cation concentration and species on the folding free energy of five large (49-124 nt) RNA hairpins, including HIV-1 TAR and molecules approximating A·U and G·C homopolymers. RNA secondary structure thermodynamics are accurately described by a model consisting of nearest-neighbor interactions and additive loop and bulge terms. Melting of small (<15 bp) duplexes and hairpins in 1M NaCl has been used to determine the parameters of this model, which is now used extensively to predict structure and folding dynamics. Few systematic measurements have been made in other ionic conditions or for larger structures. By applying mechanical force, we measured the work required to fold and unfold single hairpins at room temperature over a range of cation concentrations from 50 to 1000 mM. Free energies were then determined using the Crooks Fluctuation Theorem. We observed the following: 1. In most cases, the nearest neighbor model accurately predicted the free energy of folding at 1M NaCl. 2. Free energy was proportional to the logarithm of salt concentration. 3. Substituting potassium ions for sodium slightly decreased hairpin stability. The TAR hairpin also misfolded nearly twice as often in KCl, indicating a differential kinetic response. 4. Monovalent cation concentration affects RNA stability in a sequence-dependent manner. G·C helices were unaffected by changing salt concentration, A·U helices were modestly affected, and the hairpin loop was very sensitive. Surprisingly, the UCU bulge of TAR was found to be equally stable in all conditions tested. We also report a new estimate for the elastic parameters of single-stranded RNA.
PMCID: PMC2528546  PMID: 17997555
12.  Effects of Ligand Binding on the Mechanical Properties of Ankyrin Repeat Protein Gankyrin 
PLoS Computational Biology  2013;9(1):e1002864.
Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress. The effect is specific to those repeats of Gankyrin directly in contact with S6-C, and the mechanical ‘hot spots’ of the interaction map to the same repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein's mechanical behavior, including the order of repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.
Author Summary
Here we use molecular dynamics simulation to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with binding partner S6-C. Tandem repeat proteins like Gankyrin comprise tandem arrays of small structural motifs that pack linearly to produce elongated architectures. They are elastic, mechanically weak molecules and they unfold and refold repeat by repeat under force. We show that S6-C binding greatly increases the resistance of Gankyrin to mechanical stress. The enhanced mechanical stability is specific to those ankyrin repeats in contact with S6-C, and the localized nature of the effect results in fundamental changes in the way the protein responds to force. Thus, the forced unfolding of isolated Gankryin involves a diverse set of pathways with a preference for a C- to N-terminus unfolding mechanism whereas this diversity is reduced upon complex formation with the central repeats, which are those most tightly bound to the ligand, tending to unfold last. Our study shows how stepwise unfolding can buffer repeat proteins and their binding partners from mechanical stress in the cell. It also points to a mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.
PMCID: PMC3547791  PMID: 23341763
13.  Effect of nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine, Spermine and PAPA NONOate on intracellular pH in cardiomyocytes 
Previous studies have suggested that exogenous nitric oxide (NO) and NO-dependent signalling pathways modulate intracellular pH (pHi) in different cell types, but the role of NO in pHi regulation in the heart is poorly understood. Therefore, in this study we investigated the effect of NO donors S-nitroso-N-acetyl-DL-penicillamine, Spermine and PAPA NONOate on pHi in isolated rat ventricular myocytes.The cells were isolated from the hearts of adult Wistar rats, and pHi was monitored using a pH-sensitive fluorescent indicator 5-(and-6)-carboxy SNARF-1 with a confocal microscope. To test the effect of NO donors on sodium-hydrogen exchanger, basal pHi in Na+-free buffer and pHi recovery from intracellular acidosis after an ammonium chloride prepulse were monitored. The role of carbonic anhydrase was tested using acetazolamide. Cl−-OH− and Cl−-HCO3− exchangers were inhibited with 4,4 diisothiocyanatostilbene 2,2' disulfonic acid.All three NO donors acutely decreased pHi. This effect lasted until NO donor was removed. In a Na+-free buffer decrease in basal pHi was increased, while inhibition of carbonic anhydrase and Cl−-OH− and Cl−-HCO3− exchangers did not change the effect of NO donors on pHi. After an ammonium preload, pHi recovery was accelerated in the presence of NO donors.In conclusion, exogenous NO decreased the basal pHi leading to increased activity of sodium-hydrogen exchanger. Carbonic anhydrase and chloride-dependent sarcolemmal HCO3− and OH− transporters are not involved in the NO-induced pHi decrease in isolated rat ventricular myocytes.
PMCID: PMC3430815  PMID: 22703333
Nitric oxide; intracellular pH; cardiac myocytes; sodium hydrogen exchanger; carbonic anhydrase
14.  FTIR Studies of Collagen Model Peptides: Complementary Experimental and Simulation Approaches to Conformation and Unfolding 
X-ray crystallography of collagen model peptides has provided high resolution structures of the basic triple-helical conformation and its water-mediated hydration network. Vibrational spectroscopy provides a useful bridge for transferring the structural information from x-ray diffraction to collagen in its native environment. The vibrational mode most useful for this purpose is the Amide I mode (mostly peptide bond C=O stretch) near 1650 cm−1. The current study refines and extends the range of utility of a novel simulation method that accurately predicts the IR Amide I spectral contour from the three dimensional structure of a protein or peptide. The approach is demonstrated through accurate simulation of the experimental Amide I contour in solution for both a standard triple-helix, (Pro-Pro-Gly)10, and a second peptide with a Gly → Ala substitution in the middle of the chain that models the effect of a mutation in the native collagen sequence. Monitoring the major Amide I peak as a function of temperature gives sharp thermal transitions for both peptides, similar to those obtained by circular dichroism spectroscopy, and the FTIR spectra of the unfolded states were compared with polyproline II.
The simulation studies were extended to model early stages of thermal denaturation of (Pro-Pro-Gly)10. Dihedral angle changes suggested by molecular dynamics simulations were made in a stepwise fashion to generate peptide unwinding from each end, which emulates the effect of increasing temperature. Simulated bands from these new structures were then compared to the experimental bands obtained as temperature was increased. The similarity between the simulated and experimental IR spectra lends credence to the simulation method, and paves the way for a variety of applications.
PMCID: PMC2570338  PMID: 17550251
15.  Hemoglobin stability: observations on the denaturation of normal and abnormal hemoglobins by oxidant dyes, heat, and alkali 
Journal of Clinical Investigation  1970;49(12):2369-2376.
Several unstable mutant hemoglobins have alterations which affect areas of the molecule involved in the attachment of heme to globin. Loss of heme from globin has been demonstrated during the denaturation of some of these unstable mutants. The importance of heme ligands for the stability of hemoglobin was illustrated in the present experiments on the denaturation of several hemoglobins and hemoglobin derivatives by heat, oxidative dyes, and alkali. Heating of normal hemolysates diluted to 4 g of hemoglobin per 100 ml at 50°C for 20 hr in 0.05 M sodium phosphate, pH 7.4, caused precipitation of 23-54% of the hemoglobin. Dialysis against water or dilution of the sample decreased denaturation to 12-20%. Precipitation was decreased to less than 3.5% by the presence of 0.015 M potassium cyanide. Increasing the ionic strength of the medium increased precipitation. Cyanide prevented the formation of inclusion bodies when red cells containing unstable hemoglobin Philly, β35 tyr → phe, were incubated with the redox dye new methylene blue. Conversion to methemoglobin increased the rate of alkali denaturation of hemoglobin but the presence of potassium cyanide returned the denaturation rate to that of ferrohemoglobin. The ability of cyanide to decrease heat precipitation of hemoglobin may depend on a dimeric or tetrameric state of the hemoglobin molecule. Purified β-chains, which exist as tetramers, were stabilized but purified monomeric α-chains were not rendered more heat resistant by the ligand. Stabilization of hemoglobin by cyanide required binding of the ligand to only one heme of an αβ-dimer. Hemoglobin Gun Hill, an unstable molecule with heme groups present only on the α-chains was quite heat stable in the presence of cyanide. The binding of cyanide to the iron atom in methemoglobin is thought to be associated with increased planarity of the heme group and increased stability of the heme-globin complex. The stabilizing effect of cyanide in the above experiments suggests that Heinz body formation, heat precipitation of hemoglobin, and the increased alkali denaturation of methemoglobin depend on changes of heme-globin binding.
PMCID: PMC322738  PMID: 5480860
16.  Effects of urea on the microstructure and phase behavior of aqueous solutions of polyoxyethylene surfactants 
Membrane proteins are made soluble in aqueous buffers by the addition of various surfactants (detergents) to form so-called protein-detergent complexes (PDCs). Properties of membrane proteins are commonly assessed by unfolding the protein in the presence of surfactant in a buffer solution by adding urea. The stability of the protein under these conditions is then monitored by biophysical methods such as fluorescence or circular dichroism spectroscopy. Often overlooked in these experiments is the effect of urea on the phase behavior and micellar microstructure of the different surfactants used to form the PDCs. Here the effect of urea on five polyoxyethylene surfactants – n-octylytetraoxyethylene (C8E4), n-octylpentaoxyethylene (C8E5), n-decylhexaoxyethylene (C10E6), n-dodecylhexaoxyethylene (C12E6) and n-dodecyloctaoxylethylene (C12E8) – is explored. The presence of urea increases the critical micelle concentration (CMC) of all surfactants studied, indicating that the concentration of both the surfactant and urea should be considered in membrane protein folding studies. The cloud point temperature of all surfactants studied also increases with increasing urea concentration. Small-angle neutron scattering shows a urea-induced transition from an elongated to a globular shape for micelles of C8E4 and C12E6. In contrast, C8E5 and C12E8 form more globular micelles at room temperature and the micelles remain globular as the urea concentration is increased. The effects of increasing urea concentration on micelle structure are analogous to those of decreasing the temperature. The large changes in micelle structure observed here could also affect membrane protein unfolding studies by changing the structure of the PDC.
PMCID: PMC3045059  PMID: 21359094
17.  Long-lasting inhibition of presynaptic metabolism and neurotransmitter release by protein S-nitrosylation 
Free radical biology & medicine  2010;49(5):757-769.
Nitric oxide (NO) and related reactive nitrogen species (RNS) play a major role in the pathophysiology of stroke and other neurodegenerative diseases. One of the poorly understood consequences of stroke is a long-lasting inhibition of synaptic transmission. In this study, we tested the hypothesis that RNS can produce long-term inhibition of neurotransmitter release via S-nitrosylation of proteins in presynaptic nerve endings. We examined the effects of exogenous sources of RNS on the vesicular and non-vesicular L-[3H]glutamate release from rat brain synaptosomes. NO/RNS donors, such as spermine NONOate, MAHMA NONOate, S-nitroso-L-cysteine, and SIN-1, inhibited only the vesicular component of glutamate release with the order of potency that closely matched levels of protein S-nitrosylation. Inhibition of glutamate release persisted for >1 hr after RNS donor decomposition and wash out, and strongly correlated with decreases in the intrasynaptosomal ATP levels. Post-NO treatment of synaptosomes with thiol-reducing reagents decreased the total content of S-nitrosylated proteins but had little effect on glutamate release and ATP levels. In contrast, post-NO application of the end-product of glycolysis pyruvate partially rescued neurotransmitter release and ATP production. These data suggest that RNS suppress presynaptic metabolism and neurotransmitter release via poorly reversible modifications of glycolytic and mitochondrial enzymes, one of which was identified as glyceraldehyde-3-phosphate dehydrogenase. Similar mechanism may contribute to the long-term suppression of neuronal communication during nitrosative stress in vivo.
PMCID: PMC2923826  PMID: 20633346
nitric oxide; S-nitroso-L-cysteine; S-nitrosylation; S-nitrosation; neurotransmitter release; energetic metabolism; brain
18.  A Study of the Hydration of the Alkali Metal Ions in Aqueous Solution 
Inorganic Chemistry  2011;51(1):425-438.
The hydration of the alkali metal ions in aqueous solution has been studied by large angle X-ray scattering (LAXS) and double difference infrared spectroscopy (DDIR). The structures of the dimethyl sulfoxide solvated alkali metal ions in solution have been determined to support the studies in aqueous solution. The results of the LAXS and DDIR measurements show that the sodium, potassium, rubidium and cesium ions all are weakly hydrated with only a single shell of water molecules. The smaller lithium ion is more strongly hydrated, most probably with a second hydration shell present. The influence of the rubidium and cesium ions on the water structure was found to be very weak, and it was not possible to quantify this effect in a reliable way due to insufficient separation of the O–D stretching bands of partially deuterated water bound to these metal ions and the O–D stretching bands of the bulk water. Aqueous solutions of sodium, potassium and cesium iodide and cesium and lithium hydroxide have been studied by LAXS and M–O bond distances have been determined fairly accurately except for lithium. However, the number of water molecules binding to the alkali metal ions is very difficult to determine from the LAXS measurements as the number of distances and the temperature factor are strongly correlated. A thorough analysis of M–O bond distances in solid alkali metal compounds with ligands binding through oxygen has been made from available structure databases. There is relatively strong correlation between M–O bond distances and coordination numbers also for the alkali metal ions even though the M–O interactions are weak and the number of complexes of potassium, rubidium and cesium with well-defined coordination geometry is very small. The mean M–O bond distance in the hydrated sodium, potassium, rubidium and cesium ions in aqueous solution have been determined to be 2.43(2), 2.81(1), 2.98(1) and 3.07(1) Å, which corresponds to six-, seven-, eight- and eight-coordination. These coordination numbers are supported by the linear relationship of the hydration enthalpies and the M–O bond distances. This correlation indicates that the hydrated lithium ion is four-coordinate in aqueous solution. New ionic radii are proposed for four- and six-coordinate lithium(I), 0.60 and 0.79 Å, respectively, as well as for five- and six-coordinate sodium(I), 1.02 and 1.07 Å, respectively. The ionic radii for six- and seven-coordinate K+, 1.38 and 1.46 Å, respectively, and eight-coordinate Rb+ and Cs+, 1.64 and 1.73 Å, respectively, are confirmed from previous studies. The M–O bond distances in dimethyl sulfoxide solvated sodium, potassium, rubidium and cesium ions in solution are very similar to those observed in aqueous solution.
The hydration of alkali metal ions has been studied by large angle X-ray scattering, LAXS, and double difference infrared spectroscopy. The obtained M−O bond distances from LAXS have been compared to relevant crystal structures, conclusions about hydration numbers in aqueous solution have been made, and new ionic radii have been proposed. Hydration numbers of six, seven, eight and eight are proposed for the sodium, potassium, rubidium and cesium ions in aqueous solution.
PMCID: PMC3250073  PMID: 22168370
19.  Replication Protein A Unfolds G-Quadruplex Structures with a Varying Degree of Efficiency 
The journal of physical chemistry. B  2012;116(19):5588-5594.
Replication Protein A (RPA) is known to interact with G-rich sequences that adopt G-quadruplex (GQ) structures. Most studies in the literature have been performed on GQ formed by homogenous sequences, such as the human telomeric repeat, and RPA’s ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ forming DNA sequences (PQS) to their stability against RPA mediated unfolding using single molecule FRET and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two located in the promoter region of tyrosine hydroxylase gene are highly heterogeneous sequences, which better represent PQS in the genome. The three GQ constructs have thermal stabilities that are significantly different from each other. Our measurements showed that the most thermally stable structure (Tm= 86 °C) was also the most stable against RPA mediated unfolding, although the least thermally stable structure (Tm= 69 °C) had at least an order of magnitude higher stability against RPA mediated unfolding compared to the structure with intermediate thermal stability (Tm= 78 °C). The significance of this observation becomes more evident when considered within the context of cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these, we conclude that thermal stability is not necessarily an adequate criterion for predicting physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single molecule stopped-flow type method. All three GQ structures were unfolded within Δt≈0.30±0.10 sec, a surprising result as the unfolding time does not correlate with thermal stability or stability against RPA mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.
PMCID: PMC3434287  PMID: 22500657
20.  Hemoglobin Interactions with αB Crystallin: A Direct Test of Sensitivity to Protein Instability 
PLoS ONE  2012;7(7):e40486.
As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.
PMCID: PMC3399823  PMID: 22815750
21.  Effects of polyamines on the thermal stability and formation kinetics of DNA duplexes with abnormal structure 
Nucleic Acids Research  2001;29(24):5121-5128.
The effects of ions (i.e. Na+, Mg2+ and polyamines including spermidine and spermine) on the stability of various DNA oligonucleotides in solution were studied. These synthetic DNA molecules contained sequences that mimic various cellular DNA structures, such as duplexes, bulged loops, hairpins and/or mismatched base pairs. Melting temperature curves obtained from the ultraviolet spectroscopic experiments indicated that the effectiveness of the stabilization of cations on the duplex formation follows the order of spermine > spermidine > Mg2+ > Na+ > Tris–HCl buffer alone at pH 7.3. Circular dichroism spectra showed that salts and polyamines did not change the secondary structures of those DNA molecules under study. Surface plasmon resonance (SPR) observations suggested that the rates of duplex formation are independent of the kind of cations used or the structure of the duplexes. However, the rate constants of DNA duplex dissociation decrease in the same order when those cations are involved. The enhancement of the duplex stability by polyamines, especially spermine, can compensate for the instability caused by abnormal structures (e.g. bulged loops, hairpins or mismatches). The effects can be so great as to make the abnormal DNAs as stable as the perfect duplex, both kinetically and thermodynamically. Our results may suggest that the interconversion of various DNA structures can be accomplished readily in the presence of polyamine. This may be relevant in understanding the role of DNA polymorphism in cells.
PMCID: PMC97540  PMID: 11812845
22.  Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry, Circular Dichroism, and Turbidity Measurements 
PLoS ONE  2014;9(12):e115877.
Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).
PMCID: PMC4280130  PMID: 25548918
23.  Thermal and chemical unfolding and refolding of a eukaryotic sodium channel 
Biochimica et Biophysica Acta  2009;1788(6):1279-1286.
Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with “denatured” channels, retaining significant helical secondary structure even at 77 °C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes.
PMCID: PMC2688679  PMID: 19232514
CD, circular dichroism; DDM, dodecyl maltoside; TTX, tetrodotoxin; VGSC, voltage-gated sodium channel; cmc, critical micelle concentration; Voltage-gated sodium channel; Protein folding; Membrane protein; Secondary structure; Circular dichroism spectroscopy; Toxin binding
24.  Stable Single α-Helices Are Constant Force Springs in Proteins* 
The Journal of Biological Chemistry  2014;289(40):27825-27835.
Background: Single α-helix (SAH) domains bridge two functional domains in proteins. Their force response is poorly understood.
Results: Modeling and experiments show that SAH domains unfold non-cooperatively at low forces and maintain an approximately constant force as they unfold.
Conclusion: SAH domains act as constant force springs.
Significance: SAH domains are important mechanical elements in proteins.
Single α-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (<30 pN) without a characteristic unfolding peak. Molecular dynamics simulations showed that the SAH domain unfolds progressively as the length is increased and refolds progressively as the length is reduced. This enables the SAH domain to act as a constant force spring in the mechanically dynamic environment of the cell.
PMCID: PMC4183817  PMID: 25122759
Atomic Force Microscopy (AFM); Circular Dichroism (CD); Cytoskeleton; Molecular Dynamics; Structural Biology; Single α-Helices
25.  Electrolyte-Labile Increase of Oxygen Affinity during In Vivo Aging of Hemoglobin* 
Journal of Clinical Investigation  1967;46(10):1579-1588.
Normal human erythrocytes were separated according to in vivo age by ultracentrifugation. The “young” and “old” erythrocytes had mean cell ages of approximately 40 and 79 days, respectively. “Young” erythrocytes had a lower oxygen affinity and a higher heme-heme interaction than did “old” erythrocytes. This indicates an impairment of the oxygen-carrying function of erythrocyte hemoglobin with age.
“Young” and “old” erythrocytes were hemolyzed yielding “young” and “old” hemoglobins. “Young” hemoglobin had a comparably lower oxygen affinity than did “old” hemoglobin when the hemolysates were dialyzed against electrolyte-free water.
Exposure to sodium chloride completely obliterated this difference between the oxygen affinities and buffer values of “young” and “old” free hemoglobin. Similar exposure to potassium chloride resulted in partial obliteration of the difference between the oxygen affinities of “young” and “old” hemoglobin. Subsequent removal of sodium chloride by dialysis did not restore the pre-electrolyte differences between the oxygen affinities of “young” and “old” hemoglobin.
This evidence indicates that in vivo aging is accompanied by a conformational change of the hemoglobin molecule, which is probably due to an alteration of electrostatic interactions involving the hemoglobin molecule and which is retained after hemolysis and dialysis against water but is obliterated by addition of electrolyte. It is not possible, however, to decide from the available evidence whether this molecular change occurs independently or as a result of influences by other substances, such as 2,3-diphosphoglycerate, which also change during in vivo aging of the erythrocyte.
PMCID: PMC292906  PMID: 6061735

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