PubMed Health⌕ Search

Biomedical subjects

Bertrand García-Moreno E

Publications and source records attributed to Bertrand García-Moreno E.

12 recordsLinked to original sources
PubMed2006

High apparent dielectric constant inside a protein reflects structural reorganization coupled to the ionization of an internal Asp.

The dielectric properties of proteins are poorly understood and difficult to describe quantitatively. This limits the accuracy of methods for structure-based calculation of electrostatic energies and pK(a) values. The pK(a) values of many internal groups report apparent protein dielectric constants of 10 or higher. These values are substantially higher than the dielectric constants of 2-4 measured experimentally with dry proteins. The structural origins of these high apparent dielectric constants are not well understood. Here we report on structural and equilibrium thermodynamic studies of the effects of pH on the V66D variant of staphylococcal nuclease. In a crystal structure of this protein the neutral side chain of Asp-66 is buried in the hydrophobic core of the protein and hydrated by internal water molecules. Asp-66 titrates with a pK(a) value near 9. A decrease in the far UV-CD signal was observed, concomitant with ionization of this aspartic acid, and consistent with the loss of 1.5 turns of alpha-helix. These data suggest that the protein dielectric constant needed to reproduce the pK(a) value of Asp-66 with continuum electrostatics calculations is high because the dielectric constant has to capture, implicitly, the energetic consequences of the structural reorganization that are not treated explicitly in continuum calculations with static structures.

Aspartic Acid↗
PubMed2006

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding.

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.

Acids↗
PubMed2005

Local conformational fluctuations can modulate the coupling between proton binding and global structural transitions in proteins.

Local conformational fluctuations in proteins can affect the coupling between ligand binding and global structural transitions. This finding was established by monitoring quantitatively how the population distribution in the ensemble of microstates of staphylococcal nuclease was affected by proton binding. Analysis of acid unfolding and proton-binding data with an ensemble-based model suggests that local fluctuations: (i) can be effective modulators of ligand-binding affinities, (ii) are important determinants of the cooperativity of ligand-driven global structural transitions, and (iii) are well represented thermodynamically as local unfolding processes. These studies illustrate how an ensemble-based description of proteins can be used to describe quantitatively the interdependence of local conformational fluctuations, ligand-binding processes, and global structural transitions. This level of understanding of the relationship between conformation, energy, and dynamics is required for a detailed mechanistic understanding of allostery, cooperativity, and other complex functional and regulatory properties of macromolecules.

Binding Sites↗
PubMed2004

Stabilization of internal charges in a protein: water penetration or conformational change?

The ionizable amino acid side chains of proteins are usually located at the surface. However, in some proteins an ionizable group is embedded in an apolar internal region. Such buried ionizable groups destabilize the protein and may trigger conformational changes in response to pH variations. Because of the prohibitive energetic cost of transferring a charged group from water to an apolar medium, other stabilizing factors must be invoked, such as ionization-induced water penetration or structural changes. To examine the role of water penetration, we have measured the 17O and 2H magnetic relaxation dispersions (MRD) for the V66E and V66K mutants of staphylococcal nuclease, where glutamic acid and lysine residues are buried in predominantly apolar environments. At neutral pH, where these residues are uncharged, we find no evidence of buried water molecules near the mutation site. This contrasts with a previous cryogenic crystal structure of the V66E mutant, but is consistent with the room-temperature crystal structure reported here. MRD measurements at different pH values show that ionization of Glu-66 or Lys-66 is not accompanied by penetration of long-lived water molecules. On the other hand, the MRD data are consistent with a local conformational change in response to ionization of the internal residues.

Amino Acid Substitution↗
PubMed2003

Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects.

Single site mutations that reverse or neutralize a surface charge were made at 22 ionizable residues in staphylococcal nuclease. Unfolding free energies were obtained by guanidine hydrochloride denaturation. These data, in conjunction with previously obtained stabilities of the corresponding alanine mutants, unequivocally show that the dominant contribution to stability for virtually all of the wild-type side chains examined is the electrostatic effect associated with each residue's charged group. With only a few exceptions, these charges stabilize the native state, with an average loss of 0.5 kcal/mol of stability upon neutralization of a charge. When the charge is reversed, the average destabilization is doubled. Structure-based calculations of electrostatic free energy with the continuum method based on the finite difference solution to the linearized Poisson-Boltzmann equation reproduce the observed energetics when the polarizability in the protein interior is represented with a dielectric constant of 20. However, in some cases, large differences are found, giving insight into possible areas for improvement of the calculations. In particular, it appears that the assumptions made in the calculations about the absence of electrostatic interactions in the denatured state and the energetic consequences of dynamic fluctuations in the native state will have to be further explored.

Amino Acid Substitution↗
PubMed2002

Electrostatic effects in highly charged proteins: salt sensitivity of pKa values of histidines in staphylococcal nuclease.

The pK(a) values of most histidines in small peptides and in myoglobin increase on average by 0.30 unit between 0.02 and 1.5 M NaCl [Kao et al. (2000) Biophys. J. 79, 1637]. The DeltapK(a) values reflect primarily the ionic strength dependence of the solvation energy; screening of Coulombic interactions contributes only in a minor way. This implies that Coulombic interactions are weak, or that attractive and repulsive contributions to the pK(a) values are balanced. To distinguish experimentally between these two possibilities, and to further characterize the magnitude and salt sensitivity of surface electrostatic interactions in proteins, the salt dependence of pK(a) values of histidines in staphylococcal nuclease was measured by (1)H NMR spectroscopy. Three of the four histidines titrated with significantly depressed pK(a) values, and the salt sensitivity of all histidine pK(a) values was substantial. In three cases, the pK(a) values increased by a full unit between 0.01 and 1.5 M KCl. Anion-specific effects were found; the pK(a) values measured under equivalent ionic strengths in SCN(-) and SO(4)(2-) were higher than in Cl(-); the order of the sensitivity of pK(a) values to anions was SCN(-) > Cl(-) > SO(4)(2-). Structure-based pK(a) calculations with continuum methods were performed to interpret the measured effects structurally and to test their ability to capture the experimental behavior. Calculations in which the protein interior was treated empirically with a dielectric constant of 20 reproduced the pK(a) values and their dependence on the concentration of Cl(-). According to the calculations, the pK(a) values are depressed because of unfavorable self-energies and repulsive Coulombic interactions. Their striking salt sensitivity reflects screening of weak, repulsive, Coulombic interactions among charges separated by more than 10 A. Long-range Coulombic interactions on the surfaces of proteins are weak, but they can add up to produce substantial electrostatic effects when positive and negative charges are not balanced.

Animals↗
PubMed2002

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration.

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.

Binding Sites↗
PubMed2002

Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects.

Electrostatic contributions to the conformational stability of apoflavodoxin were studied by measurement of the proton and salt-linked stability of this highly acidic protein with urea and temperature denaturation. Structure-based calculations of electrostatic Gibbs free energy were performed in parallel over a range of pH values and salt concentrations with an empirical continuum method. The stability of apoflavodoxin was higher near the isoelectric point (pH 4) than at neutral pH. This behavior was captured quantitatively by the structure-based calculations. In addition, the calculations showed that increasing salt concentration in the range of 0 to 500 mM stabilized the protein, which was confirmed experimentally. The effects of salts on stability were strongly dependent on cationic species: K(+), Na(+), Ca(2+), and Mg(2+) exerted similar effects, much different from the effect measured in the presence of the bulky choline cation. Thus cations bind weakly to the negatively charged surface of apoflavodoxin. The similar magnitude of the effects exerted by different cations indicates that their hydration shells are not disrupted significantly by interactions with the protein. Site-directed mutagenesis of selected residues and the analysis of truncation variants indicate that cation binding is not site-specific and that the cation-binding regions are located in the central region of the protein sequence. Three-state analysis of the thermal denaturation indicates that the equilibrium intermediate populated during thermal unfolding is competent to bind cations. The unusual increase in the stability of apoflavodoxin at neutral pH affected by salts is likely to be a common property among highly acidic proteins.

Apoproteins↗
PubMed2002

Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein.

Histidine pK(a) values were measured in charge-reversal (K78E, K97E, K127E, and K97E/K127E) and charge-neutralization (E10A, E101A, and R35A) mutants of staphylococcal nuclease (SNase) by (1)H-NMR spectroscopy. Energies of interaction between pairs of charges (DeltaG(ij)) were obtained from the shifts in pK(a) values relative to wild-type values. The data describe the distance dependence and salt sensitivity of pairwise coulombic interactions. Calculations with a continuum electrostatics method captured the experimental DeltaG(ij) when static structures were used and when the protein interior was treated empirically with a dielectric constant of 20. The DeltaG(ij) when r(ij) < or = 10 A were exaggerated slightly in the calculations. Coulomb's law with a dielectric constant near 80 and a Debye-Hückel term to account for screening by the ionic strength reproduced the salt sensitivity and distance dependence of DeltaG(ij) as well as the structure-based method. In their interactions with each other, surface charges behave as if immersed in water; the Debye length describes realistically the distance where interactions become negligible at a given ionic strength. On average, charges separated by distances (r(ij)) approximately 5 A interacted with DeltaG(ij) approximately 0.6 kcal/mole in 0.01 M KCl, but DeltaG(ij) decayed to < or =0.10 kcal/mole when r(ij) = 20 A. In 0.10 M KCl, DeltaG(ij) approximately 0.10 kcal/mole when r(ij) = 10 A. In 1.5 M KCl, only short-range interactions with r(ij) < or = 5 A persisted. Although at physiological ionic strengths the interactions between charges separated by more than 10 A are extremely weak, in situations where charge imbalance exists many weak interactions can cumulatively produce substantial effects.

Histidine↗
PubMedDate not supplied

Human monoclonal antibody stability and activity at vaginal pH.

Antibodies can be delivered topically to the vagina to protect against pregnancy and sexually transmitted infections, but the acidity of vaginal secretions (pH 3.5-4.5) might inactivate them. To address this question, both experimental and computational methods were used to evaluate the effects of pH on human monoclonal antibody (MAb) stability and activity. To determine the acid-sensitivity of their antigen binding sites, human MAbs against human sperm (H6-3C4) and gp120 of HIV (1511) were tested by ELISA for binding to human sperm and recombinant gp120, respectively, at pH 3.0-7.0, after storing them for 1 or 20 h at the same pH. Binding was unaltered by acidic pH> or =4 even after 20 h, and at pH 3.5 both MAbs retained > or =40% antigen binding activity. A humanized MAb against HSV-2 glycoprotein B expressed both in Chinese hamster ovary (CHO) cells and in soybean cells was incubated for 1 or 24 h at pH 3.5-7.6, brought to neutral pH, and tested for ability to block HSV-2 infection of foreskin fibroblast cells. Loss in blocking activity occurred only when antibodies were incubated at pH 3.5 for 24 h and was independent of the expression cell type. Using empirical structure-based methods, net charge, Z, and electrostatic contributions to free energy, DeltaDeltaG(el), were calculated as a function of pH for 1 human and 8 murine F(ab)s. The calculations indicate that Z changes slowly between pH 5.0 and 9.0 and that DeltaDeltaG(el) is nearly constant between pH 4.0 and 10 for all the F(ab)s and, therefore, human antibodies should remain stable in this pH range. Taken together, our data and empirical calculations suggest that vaginally applied human MAbs are likely to remain stable and active throughout the duration they are likely to reside in the vagina.

Antibodies, Monoclonal↗