RESUMO
It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.
Assuntos
Desnaturação Proteica , Dobramento de Proteína , Resposta a Proteínas não Dobradas/fisiologia , Substituição de Aminoácidos , Fenômenos Biofísicos , Cristalografia por Raios X , Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Pressão , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Solventes , Espectrometria de Fluorescência , Triptofano/química , Água/químicaRESUMO
Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK(a) values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK(a) values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK(a); they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments.
Assuntos
Arginina/química , Nuclease do Micrococo/química , Modelos Moleculares , Conformação Proteica , Guanidina/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Eletricidade Estática , Termodinâmica , Água/químicaRESUMO
Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK(a) values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK(a) values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H(+) transport. The pK(a) values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK(a) values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.
Assuntos
Íons/química , Lisina/química , Nuclease do Micrococo/química , Estabilidade Enzimática , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Conformação Proteica , TermodinâmicaRESUMO
The effects of cavity-creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C-terminal domain and the interface between α and ß subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the ß-barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions.
Assuntos
Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Mutação Puntual , Dobramento de Proteína , Staphylococcus/enzimologia , Nuclease do Micrococo/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Estabilidade Proteica , Staphylococcus/química , Staphylococcus/genética , TermodinâmicaRESUMO
The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information.
Assuntos
Nuclease do Micrococo/química , Ressonância Magnética Nuclear Biomolecular/métodos , Dobramento de Proteína , Staphylococcus/enzimologia , Cinética , Nuclease do Micrococo/genética , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Staphylococcus/química , Staphylococcus/genéticaRESUMO
The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the Δ+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of Δ+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Δ+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.
Assuntos
Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Substituição de Aminoácidos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de ProteínaRESUMO
The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of protein electrostatics. To improve structure-based energy calculations, it is necessary to better understand the physical character and molecular determinants of electrostatic effects. Thus, the Cooperative intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods, the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pK(a) values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pK(a) values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pK(a) values in water. Many computational methods were tested in this first Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to objectively assess the performance of many computational methods tested on this one extensive data set. This volume of Proteins: Structure, Function, and Bioinformatics introduces the pK(a) Cooperative, presents reports submitted by participants in the Blind Prediction Challenge, and highlights some of the problems in structure-based calculations identified during this exercise.
Assuntos
Simulação por Computador , Proteínas/química , Proteínas/metabolismo , Ácidos/química , Concentração de Íons de Hidrogênio , Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Modelos Químicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pesquisa , Eletricidade Estática , Estatística como AssuntoRESUMO
Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral and moved toward crevices and toward the protein-water interface when they were charged. The only exceptions are Lys-36, Lys-62, and Lys-103, which remained buried even after charging. With the exception of Lys-38, the number of internal water molecules surrounding the ionizable group increased upon charging: the average number of water oxygen atoms within the first hydration shell increased by 1.7 for Lys residues, by 5.2 for Asp residues, and by 3.2 for Glu residues. The polarity of the microenvironment of the ionizable group also increased when the groups were charged: the average number of polar atoms of any kind within the first hydration shell increased by 2.7 for Lys residues, by 4.8 for Asp residues, and by 4.0 for Glu residues. An unexpected correlation was observed between the absolute value of the shifts in pK(a) values measured experimentally, and several parameters of structural relaxation: the net difference in the polarity of the microenvironment of the charged and neutral forms of the ionizable groups, the net difference in hydration of the charged and neutral forms of the ionizable groups, and the difference in RMSD values of the charged and neutral forms of the ionizable groups. The effects of ionization of internal groups on the conformation of the backbone were noticeable but mostly small and localized to the area immediately next to the internal ionizable moiety. Some variants did exhibit local unfolding.
Assuntos
Nuclease do Micrococo/química , Água/química , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica MolecularRESUMO
Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK(a) values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.
Assuntos
Aminoácidos/química , Nuclease do Micrococo/química , Ribonuclease H/química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Íons , Modelos Moleculares , Proteínas Mutantes/química , Conformação Proteica , TermodinâmicaRESUMO
A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.
Assuntos
Pressão Atmosférica , Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Desdobramento de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Varredura Diferencial de Calorimetria , Concentração de Íons de Hidrogênio , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Solventes , TemperaturaRESUMO
The pK(a) values measured previously for the internal Lys-66, Asp-66, and Glu-66 in variants of a highly stable form of staphylococcal nuclease are shifted by as many as 5 pK(a) units relative to normal pK(a) values in water. These shifts cannot be reproduced with continuum electrostatics calculations with static structures unless the protein is treated with high dielectric constants near 10. These high apparent dielectric constants are inconsistent with the highly hydrophobic microenvironments of the ionizable moieties in crystal structures. To examine the origins of these high apparent dielectric constants, we showed that the pK(a) values of these internal residues are sensitive to the global stability of the protein; the shifts tend to be smaller in less stable forms of nuclease. This implies that the apparent dielectric constants reported by these internal ionizable groups are high because they reflect conformational reorganization coupled to their ionization. To detect this directly, acid-base titrations monitored with Trp fluorescence and near-UV and far-UV CD spectroscopy were performed on variants with Lys-66, Glu-66, or Asp-66 in background proteins with different stabilities. Conformational reorganization coupled to the ionization of the internal groups was spectroscopically detectable, especially in the less stable background proteins. The data show that to improve the accuracy of structure-based pK(a) calculations of internal groups the calculations will have to treat explicitly all structural reorganization coupled to ionization. The data also suggest a novel approach to mapping the folding free energy landscape of proteins by using internal ionizable groups to stabilize partially unfolded states.
Assuntos
Ácido Aspártico/química , Ácido Glutâmico/química , Lisina/química , Nuclease do Micrococo/química , Ácido Aspártico/genética , Dicroísmo Circular , Cristalografia por Raios X , Ácido Glutâmico/genética , Concentração de Íons de Hidrogênio , Cinética , Lisina/genética , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Conformação Proteica , TermodinâmicaRESUMO
Recent work has shown that proteins can tolerate hydrophobic-to-ionizable-residue mutations. Here, we provide experimental evidence that the essential properties (pK value, protonation state, local dynamics) of buried ionizable groups in proteins can be efficiently modulated through the rational design of the surface charge distribution, thus paving the way for the protein engineering exploitation of charge burial.
Assuntos
Íons/química , Proteínas/química , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas/genética , Eletricidade Estática , Termodinâmica , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
Prior computational studies of the acid-unfolding behavior of staphylococcal nuclease (SNase) suggest that the pK(a) values of its carboxylic groups are difficult to reproduce with electrostatics calculations with continuum methods. To examine the molecular determinants of the pK(a) values of carboxylic groups in SNase, the pK(a) values of all 20 Asp and Glu residues were measured with multidimensional and multinuclear NMR spectroscopy in an acid insensitive variant of SNase. The crystal structure of the protein was obtained to describe the microenvironments of the carboxylic groups. Fourteen Asp and Glu residues titrate with relatively normal pK(a) values that are depressed by less than 1.1 units relative to the normal pK(a) of Asp and Glu in water. Only six residues have pK(a) values shifted by more than 1.5 units. Asp-21 has an unusually high pK(a) of 6.5, which is probably the result of interactions with other carboxylic groups at the active site. The most perturbed pK(a) values appear to be governed by hydrogen bonding and not by Coulomb interactions. The pK(a) values calculated with standard continuum electrostatics methods applied to static structures are more depressed than the measured values because Coulomb effects are exaggerated in the calculations. The problems persist even when the protein is treated with the dielectric constant of water. This can be interpreted to imply that structural relaxation is an important determinant of the pK(a) values; however, no major pH-sensitive conformational reorganization of the backbone was detected using NMR spectroscopy.
Assuntos
Ácido Aspártico/química , Ácido Glutâmico/química , Nuclease do Micrococo/química , Calibragem , Cristalografia por Raios X/métodos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
We report direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein, demonstrating heteronuclear 1H-15N NMR data on the Lys-66 side chain amine (Nzeta) group in the delta-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 Nzeta group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the 1Hzeta and 15Nzeta chemical shifts at pH 8.0 and 6 degrees C for the buried lysine are 0.81 and 23.3 ppm, respectively, which are too abnormal to correspond to the protonated (NH3+) state. Further investigations using a model system suggested that the abnormal 1H and 15N chemical shifts represent the deprotonated (NH2) state of the Lys-66 Nzeta group. More straightforward evidence for the deprotonation was obtained with 2D F1-1H-coupled 1H-15N heteronuclear correlation experiments. Observed 15N multiplets clearly indicated that the spin system for the Lys-66 Nzeta group is AX2 (NH2) rather than AX3 (NH3+). Interestingly, although the amine group is buried in the hydrophobic core, the hydrogen exchange between water and the Lys-66 Nzeta group was found to be relatively rapid (93 s(-1) at -1 degrees C), which suggests the presence of a dynamic process such as local unfolding or water penetration. The partial self-decoupling effect on 15Nzeta multiplets due to the rapid hydrogen exchange is also discussed.
Assuntos
Lisina/química , Nuclease do Micrococo/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , PrótonsRESUMO
Herein, we probe by pressure perturbation calorimetry (PPC) the coefficient of thermal expansion, the volumetric and the hydration properties of variants of a hyperstable variant of staphylococcal nuclease (SNase), Delta+PHS. The temperature-dependent volumetric properties of the folded and unfolded states of the wild-type protein are calculated with previously published data. The present PPC results are used to interpret the volume diagram and expansivity at a molecular level. We conclude that the expansivity of the unfolded state is, to a first approximation, temperature independent, while that of the folded state decreases with increasing temperature. Our data suggest that at low temperature the defining contribution to DeltaV comes mainly from excluded volume differences and DeltaV for unfolding is negative. In contrast, at high temperatures, differential solvation due to the increased exposed surface area of the unfolded state and, in particular, its larger thermal volume linked to the increased conformational dynamics of the unfolded state ensemble takes over and DeltaV for unfolding eventually becomes positive.
Assuntos
Nuclease do Micrococo/química , Dobramento de Proteína , Proteínas/química , Água/química , Calorimetria , Pressão , Conformação Proteica , Desnaturação ProteicaRESUMO
The ionization properties of Lys and Glu residues buried in the hydrophobic core of staphylococcal nuclease (SN) suggest that the interior of this protein behaves as a highly polarizable medium with an apparent dielectric constant near 10. This has been rationalized previously in terms of localized conformational relaxation concomitant with the ionization of the internal residue, and with contributions by internal water molecules. Paradoxically, the crystal structure of the SN V66E variant shows internal water molecules and the structure of the V66K variant does not. To assess the structural and dynamical character of interior water molecules in SN, a series of 10-ns-long molecular dynamics (MD) simulations was performed with wild-type SN, and with the V66E and V66K variants with Glu66 and Lys66 in the neutral form. Internal water molecules were identified based on their coordination state and characterized in terms of their residence times, average location, dipole moment fluctuations, hydrogen bonding interactions, and interaction energies. The locations of the water molecules that have residence times of several nanoseconds and display small mean-square displacements agree well with the locations of crystallographically observed water molecules. Additional, relatively disordered water molecules that are not observed crystallographically were found in internal hydrophobic locations. All of the interior water molecules that were analyzed in detail displayed a distribution of interaction energies with higher mean value and narrower width than a bulk water molecule. This underscores the importance of protein dynamics for hydration of the protein interior. Further analysis of the MD trajectories revealed that the fluctuations in the protein structure (especially the loop elements) can strongly influence protein hydration by changing the patterns or strengths of hydrogen bonding interactions between water molecules and the protein. To investigate the dynamical response of the protein to burial of charged groups in the protein interior, MD simulations were performed with Glu66 and Lys66 in the charged state. Overall, the MD simulations suggest that a conformational change rather than internal water molecules is the dominant determinant of the high apparent polarizability of the protein interior.
Assuntos
Nuclease do Micrococo/química , Proteômica/métodos , Água/química , Algoritmos , Simulação por Computador , Cristalografia por Raios X , Ácido Glutâmico/química , Hidrogênio/química , Ligação de Hidrogênio , Cinética , Lisina/química , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Software , Solventes , Eletricidade Estática , Termodinâmica , Fatores de TempoRESUMO
Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of â¼ 12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.
Assuntos
Ácidos/química , Arginina/química , Proteínas/química , Sítios de Ligação , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Bicamadas Lipídicas/química , Espectroscopia de Ressonância MagnéticaRESUMO
Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK(a) calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.
Assuntos
Proteínas de Bactérias/química , Nuclease do Micrococo/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Estabilidade Enzimática , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Nuclease do Micrococo/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Desdobramento de Proteína , Staphylococcus aureus/enzimologia , TermodinâmicaRESUMO
Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.biomedcentral.com/1741-7007/7/69.
Assuntos
Adaptação Biológica/fisiologia , Membranas Intracelulares/fisiologia , Proteínas de Membrana/fisiologia , Frações Subcelulares/fisiologia , Animais , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/química , Proteínas de Membrana/química , Frações Subcelulares/químicaRESUMO
Understanding protein stabilization by small organic compounds is a topic of great practical importance. The effect of mannosylglycerate, a charged compatible solute typical of thermophilic microorganisms, on a variant of staphylococcal nuclease was investigated using several NMR spectroscopy methods. No structural changes were apparent from the chemical shifts of amide protons. Measurements of (15)N relaxation and model-free analysis, water-amide saturation transfer (phase-modulated CLEAN chemical exchange), and hydrogen/deuterium exchange rates provided a detailed picture of the effects of mannosylglycerate on the backbone dynamics and time-averaged structure of this protein. The widest movements of the protein backbone were significantly constrained in the presence of mannosylglycerate, as indicated by the average 5-fold decrease of the hydrogen/deuterium exchange rates, but the effect on the millisecond timescale was small. At high frequencies, internal motions of staphylococcal nuclease were progressively restricted with increasing concentrations of mannosylglycerate or reduced temperature, while the opposite effect was observed with urea (a destabilizing solute). The order parameters showed a strong correlation with the changes in the T(m) values induced by different solutes, determined by differential scanning calorimetry. These data show that mannosylglycerate caused a generalised reduction of backbone motions and demonstrate a correlation between protein stabilization and protein rigidification.