Continuum Electrostatic Model Research Articles

Improved pKa prediction: Combining empirical and semimicroscopic methods

Using three different methods we tried to compute 171 experimentally known pK(a) values of ionizable residues from 15 different proteins and compared the accuracies of computed pK(a) values in terms of the root mean square deviation (RMSD) from experiment. One method is based on a continuum electrostatic model of the protein including conformational flexibility (KBPLUS). The others are empirical approaches with PROPKA deploying physically motivated energy terms with adjustable parameters and PKAcal using an empirical function with no physical basis. PROPKA reproduced the pK(a) values with highest overall accuracy. Differentiating the data set into weakly and strongly shifted experimental pK(a) values, however, we found that PROPKA's accuracy is better if the pK(a) values are weakly shifted but on equal footing with that of KBPLUS for more strongly shifted values. On the other hand, PKAcal reproduces strongly shifted pK(a) values badly but weakly shifted values with the same accuracy as PROPKA. We tested different consensus approaches combining data from all three methods to find a general procedure for most accurate pK(a) predictions. In most of the cases we found that the consensus approach reproduced experimental data with better accuracy than any of the individual methods alone.

Optimized Parameters for Continuum Solvation Calculations with Carbohydrates

Continuum solvent models have been shown to be an efficient method for the calculation of the energetics of biomolecules in solution. However, for these methods to produce accurate results, an appropriate set of atomic radii or volumes is needed. While these have been developed for proteins and nucleic acids, the same is not true of carbohydrates. Here, a set of optimized parameters for continuum solvation calculations of carbohydrates in conjunction with the Carbohydrate Solution Force Field are presented. Explicit solvent free-energy perturbation simulations were performed on a set of hexapyranose sugars and used to fit atomic radii for Poisson-Boltzmann and generalized-Born calculations, and to fit atomic volumes for use with the analytical continuum electrostatics model. The solvation energetics computed with the optimized radii and a Poisson-Boltzmann model show remarkable agreement with explicit solvent simulation, with a root-mean-square error of 1.19 kcal/mol over a large test set of sugars in many conformations. The generalized-Born model gives slightly poorer agreement, but still correlates very strongly, with an error of 1.69 kcal/mol. The analytical continuum electrostatics model correlates well with the explicit solvent results, but gives a larger error of 4.71 kcal/mol. The remarkable agreement between the solvation free energies computed in explicit and implicit solvent provides strong motivation for the use of implicit solvent models in the simulation of carbohydrate-containing systems.

A Method to Determine Dielectric Constants in Nonhomogeneous Systems: Application to Biological Membranes

Continuum electrostatic models have had quantitative success in describing electrostatic-mediated phenomena on atomistic scales; however, there continues to be significant disagreement about how to assign dielectric constants in mixed, nonhomogeneous systems. We introduce a method for determining a position-dependent dielectric profile from molecular dynamics simulations. In this method, the free energy of introducing a test charge is computed two ways: from a free energy perturbation calculation and from a numerical solution to Poisson's Equation. The dielectric profile of the system is then determined by minimizing the discrepancy between these two calculations simultaneously for multiple positions of the test charge. We apply this method to determine the dielectric profile of a lipid bilayer surrounded by water. We find good agreement with dielectric models for lipid bilayers obtained by other approaches. The free energy of transferring an ion from bulk water to the lipid bilayer computed from the atomistic simulations indicates that large errors are introduced when the bilayer is represented as a single slab of low dielectric embedded in the higher-dielectric solvent. Significant improvement results from introducing an additional layer of intermediate dielectric (∼3) on each side of the low dielectric core extending from ∼12 Å to 18 Å. A small dip in transfer free energy just outside the lipid headgroups indicates the presence of a very high dielectric. These results have implications for the design of implicit membrane models and our understanding of protein-membrane interactions.

Polarizable Atomic Multipole Solutes in a Generalized Kirkwood Continuum.

The generalized Born (GB) model of continuum electrostatics is an analytic approximation to the Poisson equation useful for predicting the electrostatic component of the solvation free energy for solutes ranging in size from small organic molecules to large macromolecular complexes. This work presents a new continuum electrostatics model based on Kirkwood's analytic result for the electrostatic component of the solvation free energy for a solute with arbitrary charge distribution. Unlike GB, which is limited to monopoles, our generalized Kirkwood (GK) model can treat solute electrostatics represented by any combination of permanent and induced atomic multipole moments of arbitrary degree. Here we apply the GK model to the newly developed Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field, which includes permanent atomic multipoles through the quadrupole and treats polarization via induced dipoles. A derivation of the GK gradient is presented, which enables energy minimization or molecular dynamics of an AMOEBA solute within a GK continuum. For a series of 55 proteins, GK electrostatic solvation free energies are compared to the Polarizable Multipole Poisson-Boltzmann (PMPB) model and yield a mean unsigned relative difference of 0.9%. Additionally, the reaction field of GK compares well to that of the PMPB model, as shown by a mean unsigned relative difference of 2.7% in predicting the total solvated dipole moment for each protein in this test set. The CPU time needed for GK relative to vacuum AMOEBA calculations is approximately a factor of 3, making it suitable for applications that require significant sampling of configuration space.

Influence of the Protein Environment on the Redox Potentials of Flavodoxins from Clostridium beijerinckii

The flavin mononucleotide (FMN) quinones in flavodoxin have two characteristic redox potentials, namely, Em(FMNH./FMNH-) for the one-electron reduction of the protonated FMN (E1) and Em(FMN/FMNH.) for the proton-coupled one-electron reduction (E2). These redox potentials in native and mutant flavodoxins obtained from Clostridium beijerinckii were calculated by considering the protonation states of all titratable sites as well as the energy contributed at the pKa value of FMN during protonation at the N5 nitrogen (pKa(N5)). E1 is sensitive to the subtle differences in the protein environments in the proximity of FMN. The protein dielectric volume that prevents the solvation of charged FMN quinones is responsible for the downshift of 130-160 mV of the E1 values with respect to that in an aqueous solution. The influence of the negatively charged 5'-phosphate group of FMN quinone on E1 could result in a maximum shift of 90 mV. A dramatic difference of 130 mV in the calculated E2 values of FMN quinone of the native and G57T mutant flavodoxins is due to the difference in the pKa(N5) values. This is due to the difference in the influence exerted by the carbonyl group of the protein backbone at residue 57.

Polarizable atomic multipole solutes in a Poisson-Boltzmann continuum.

Modeling the change in the electrostatics of organic molecules upon moving from vacuum into solvent, due to polarization, has long been an interesting problem. In vacuum, experimental values for the dipole moments and polarizabilities of small, rigid molecules are known to high accuracy; however, it has generally been difficult to determine these quantities for a polar molecule in water. A theoretical approach introduced by Onsager [J. Am. Chem. Soc. 58, 1486 (1936)] used vacuum properties of small molecules, including polarizability, dipole moment, and size, to predict experimentally known permittivities of neat liquids via the Poisson equation. Since this important advance in understanding the condensed phase, a large number of computational methods have been developed to study solutes embedded in a continuum via numerical solutions to the Poisson-Boltzmann equation. Only recently have the classical force fields used for studying biomolecules begun to include explicit polarization in their functional forms. Here the authors describe the theory underlying a newly developed polarizable multipole Poisson-Boltzmann (PMPB) continuum electrostatics model, which builds on the atomic multipole optimized energetics for biomolecular applications (AMOEBA) force field. As an application of the PMPB methodology, results are presented for several small folded proteins studied by molecular dynamics in explicit water as well as embedded in the PMPB continuum. The dipole moment of each protein increased on average by a factor of 1.27 in explicit AMOEBA water and 1.26 in continuum solvent. The essentially identical electrostatic response in both models suggests that PMPB electrostatics offers an efficient alternative to sampling explicit solvent molecules for a variety of interesting applications, including binding energies, conformational analysis, and pK(a) prediction. Introduction of 150 mM salt lowered the electrostatic solvation energy between 2 and 13 kcalmole, depending on the formal charge of the protein, but had only a small influence on dipole moments.

Recognition of Ribonuclease A by 3′–5′-Pyrophosphate-Linked Dinucleotide Inhibitors: A Molecular Dynamics/Continuum Electrostatics Analysis

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5′-phospho-2′-deoxyuridine-3-pyrophosphate (P→5)-adenosine-3-phosphate (pdUppA-3′-p). The 3′,5′-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B 2 and the placement of the 5′- β-phosphate of the adenylate (instead of the α-phosphate) at subsite P 1 where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3′-p and the related weaker inhibitor dUppA, which lacks the 3′ and 5′ terminal phosphate groups of pdUppA-3′-p. The simulations show that the adenylate 5′- β-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P 1 and B 2. The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3′-p varies between −7.9 kcal/mol and −2.8 kcal/mol for a range of protein/ligand dielectric constants ( ϵ p) 2–20, in good agreement with the experimental value (−3.6 kcal/mol); the agreement becomes exact with ϵ p = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3′-p make increased interactions with residues Lys 7 and Lys 66 of the more remote sites P 2 and P 0, and His 119 of site P 1.

Molecular dynamics study of the stabilities of consensus designed ankyrin repeat proteins

Two designed ankyrin repeat (AR) proteins (E3_5 and E3_19) are high homologous (with about 87% sequence identity) and their crystal structures have a Calpha atom-positional root-mean-square difference of about 0.14 nm. However, it was found that E3_5 is considerably more stable than E3_19 in guanidinium hydrochloride and thermal denaturation experiments. With the goal of providing insights into the various factors contributing to the stabilities of the designed AR proteins and suggesting possible mutations to enhance their stabilities, homology modeling and molecular dynamics (MD) simulations with explicit solvent have been performed. Because the crystal structure of E3_19 was solved later than that of E3_5, a homology model of E3_19 based on the crystal structure of E3_5 was also used in the simulations. E3_5 shows a very stable trajectory in both crystal and solution simulations. In contrast, the C-terminal repeat of E3_19 unfolds in the simulations starting from either the modeled structure or the crystal structure, although it has a sequence identical to that of E3_5. A continuum electrostatic model was used to estimate the effect of single mutations on protein stability and to study the interaction between the internal ARs and the C-terminal capping AR. Mutations involving charged residues were found to have large effects on stability. Due to the difference in charge distribution in the internal ARs of E3_19 and E3_5, their interaction with the C-terminal capping AR is less favorable in E3_19. The simulation trajectories suggest that the stability of the designed AR proteins can be increased by optimizing the electrostatic interactions within and between the different repeats.

Molecular Dynamics Simulations Show That Bound Mg2+ Contributes to Amino Acid and Aminoacyl Adenylate Binding Specificity in Aspartyl-tRNA Synthetase through Long Range Electrostatic Interactions

Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry. The Mg2+ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP x Mg3(2+) complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg2+ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNA-bound AspRS, the single remaining Mg2+ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg2+ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states.

BALLView: a tool for research and education in molecular modeling

We present BALLView, a molecular viewer and modeling tool. It combines state-of-the-art visualization capabilities with powerful modeling functionality including implementations of force field methods and continuum electrostatics models. BALLView is a versatile and extensible tool for research in structural bioinformatics and molecular modeling. Furthermore, the convenient and intuitive graphical user interface offers novice users direct access to the full functionality, rendering it ideal for teaching. Through an interface to the object-oriented scripting language Python it is easily extensible.

H++: a server for estimating pKas and adding missing hydrogens to macromolecules

The structure and function of macromolecules depend critically on the ionization (protonation) states of their acidic and basic groups. A number of existing practical methods predict protonation equilibrium pK constants of macromolecules based upon their atomic resolution Protein Data Bank (PDB) structures; the calculations are often performed within the framework of the continuum electrostatics model. Unfortunately, these methodologies are complex, involve multiple steps and require considerable investment of effort. Our web server provides access to a tool that automates this process, allowing both experts and novices to quickly obtain estimates of pKs as well as other related characteristics of biomolecules such as isoelectric points, titration curves and energies of protonation microstates. Protons are added to the input structure according to the calculated ionization states of its titratable groups at the user-specified pH; the output is in the PQR (PDB + charges + radii) format. In addition, corresponding coordinate and topology files are generated in the format supported by the molecular modeling package AMBER. The server is intended for a broad community of biochemists, molecular modelers, structural biologists and drug designers; it can also be used as an educational tool in biochemistry courses.

Relative contributions of desolvation, inter‐ and intramolecular interactions to binding affinity in protein kinase systems

In several previous studies, we performed sensitivity analysis to gauge the relative importance of different atomic partial charges in determining protein-ligand binding. In this work, we gain further insights by decomposing these results into three contributions: desolvation, intramolecular interactions, and intermolecular interactions, again based on a Poisson continuum electrostatics model. Three protein kinase-inhibitor systems have been analyzed: CDK2-deschloroflavopiridol, PKA-PKI, and LCK-PP2. Although our results point out the importance of specific intermolecular interactions to the binding affinity, they also reveal the remarkable contributions from the solvent-mediated intramolecular interactions in some cases. Thus, it is necessary to look beyond analyzing protein-ligand interactions to understand protein-ligand recognition or to gain insights into designing ligands and proteins. In analyzing the contributions of the three components to the overall binding free energy, the PKA-PKI system with a much larger ligand was found to behave differently from the other two systems with smaller ligands. In the former case, the intermolecular interactions are very favorable, and together with the favorable solvent-mediated intramolecular interactions, they overcome the large desolvation penalties to give a favorable electrostatics contribution to the overall binding affinity. On the other hand, the other two systems with smaller ligands only present modest intermolecular interactions and they are not or are only barely sufficient to overcome the desolvation penalty even with the aid of the favorable intramolecular contributions. As a result, the binding affinity of these two systems do not or only barely benefit from electrostatics contributions.

On the role of the conserved aspartate in the hydrolysis of the phosphocysteine intermediate of the low molecular weight tyrosine phosphatase.

The usual rate-determining step in the catalytic mechanism of the low molecular weight tyrosine phosphatases involves the hydrolysis of a phosphocysteine intermediate. To explain this hydrolysis, general base-catalyzed attack of water by the anion of a conserved aspartic acid has sometimes been invoked. However, experimental measurements of solvent deuterium kinetic isotope effects for this enzyme do not reveal a rate-limiting proton transfer accompanying dephosphorylation. Moreover, base activation of water is difficult to reconcile with the known gas-phase proton affinities and solution phase pK(a)'s of aspartic acid and water. Alternatively, hydrolysis could proceed by a direct nucleophilic attack by a water molecule. To understand the hydrolysis mechanism, we have used high-level density functional methods of quantum chemistry combined with continuum electrostatics models of the protein and the solvent. Our calculations do not support a catalytic activation of water by the aspartate. Instead, they indicate that the water oxygen directly attacks the phosphorus, with the aspartate residue acting as a H-bond acceptor. In the transition state, the water protons are still bound to the oxygen. Beyond the transition state, the barrier to proton transfer to the base is greatly diminished; the aspartate can abstract a proton only after the transition state, a result consistent with experimental solvent isotope effects for this enzyme and with established precedents for phosphomonoester hydrolysis.

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study.

A continuum electrostatics model is used to calculate the relative stabilities of 117 mutants of staphylococcal nuclease (SNase) involving the mutation of a charged residue to an uncharged residue. The calculations are based on the crystallographic structure of the wild-type protein and attempt to take implicitly into account the effect of the mutations in the denatured state by assuming a linear relationship between the free energy changes caused by the mutation in the native and denatured states. A good correlation (linear correlation coefficient of approximately 0.8) is found with published experimental relative stabilities of these mutants. The results suggest that in the case of SNase (i) charged residues contribute to the stability of the native state mainly through electrostatic interactions, and (ii) native-like electrostatic interactions may persist in the denatured state. The continuum electrostatics method is only moderately sensitive to model parameters and leads to quasi-predictive results for the relative mutant stabilities (error of 2-3 kJ mol(-1) or of the order of k(B)T), except for mutants in which a charged residue is mutated to glycine.

Free energy of solvation from molecular dynamics simulations for low dielectric solvents.

Using molecular dynamics simulation, we present new results for the free energy of solvation for solvents with low dielectric constants (CCl(4), CHCl(3), benzene). The solvation free energy is computed as the sum of three contributions originated at the cavitation of the solute by the solvent, the solute-solvent repulsion and dispersion interactions, and the electrostatic solvation of the solute. The cavitational contribution has been obtained from the Claverie-Pierotti model applied to excluded volumes obtained from distances for nearest neighbor configurations between the solute's atoms and a spherical solvent description. An electrostatic continuum model has been adapted for the computation of the electrostatic free energy of solvation, whereas the van der Waals contribution has been calculated directly from the intermolecular interactions defined by the force fields applied to the simulations. For each solvent, a large set of solute molecules containing most of the chemically interesting functionalities has been treated. The simulated solvation free energies are in very good agreement with experimental data, although a small systematical overestimation of the free energy of solvation indicates a failure of the spherical approach to the solvent molecules in the case of benzene.

On the transferability of hydration-parametrized continuum electrostatics models to solvated binding calculations.

Using molecular mechanics force field partial atomic charges, we show the nonuniqueness of the parametrization of continuum electrostatics models with respect to solute atomic radii and interior dielectric constant based on hydration (vacuum-to-water transfer) free energy data available for small molecules. Moreover, parameter sets that are optimal and equivalent for hydration free energy calculations lead to large variations of calculated absolute and relative electrostatic binding free energies. Hence, parametrization of solvation effects based on hydration data, although a necessary condition, is not sufficient to guarantee its transferability to the calculation of binding free energies in solution.

Protein electrostatics: A review of the equations and methods used to model electrostatic equations in biomolecules – Applications in biotechnology

The molecular understanding of the initial interaction between a protein and, e.g., its substrate, a surface or an inhibitor is essentially an understanding of the role of electrostatics in intermolecular interactions. When studying biomolecules it is becoming increasingly evident that electrostatic interactions play a role in folding, conformational stability, enzyme activity and binding energies as well as in protein-protein interactions. In this chapter we present the key basic equations of electrostatics necessary to derive the equations used to model electrostatic interactions in biomolecules. We will also address how to solve such equations. This chapter is divided into two major sections. In the first part we will review the basic Maxwell equations of electrostatics equations called the Laws of Electrostatics that combined will result in the Poisson equation. This equation is the starting point of the Poisson-Boltzmann (PB) equation used to model electrostatic interactions in biomolecules. Concepts as electric field lines, equipotential surfaces, electrostatic energy and when can electrostatics be applied to study interactions between charges will be addressed. In the second part we will arrive at the electrostatic equations for dielectric media such as a protein. We will address the theory of dielectrics and arrive at the Poisson equation for dielectric media and at the PB equation, the main equation used to model electrostatic interactions in biomolecules (e.g., proteins, DNA). It will be shown how to compute forces and potentials in a dielectric medium. In order to solve the PB equation we will present the continuum electrostatic models, namely the Tanford-Kirkwood and the modified Tandord-Kirkwood methods. Priority will be given to finding the protonation state of proteins prior to solving the PB equation. We also present some methods that can be used to map and study the electrostatic potential distribution on the molecular surface of proteins. The combination of graphical visualisation of the electrostatic fields combined with knowledge about the location of key residues on the protein surface allows us to envision atomic models for enzyme function. Finally, we exemplify the use of some of these methods on the enzymes of the lipase family.

Accurate Transferable Model for Water,n-Octanol, andn-Hexadecane Solvation Free Energies

We present a fast continuum method for the calculation of solvation free energies. It is based on a continuum electrostatics model with MMFF94 atomic charges combined with a nonelectrostatic term, which is a linear function of the solvent-accessible surface area. The model's parameters have been optimized using sets of 410, 382, and 2116 molecules for gas−water, gas−hexadecane, and water−octanol transfer, respectively. These are the largest, most diverse sets of molecules used to date for a similar solvation model. The model's predictive power was verified by using 90% of the molecule set for training and the remainder as a test set. The average test set errors differed by only about 1% from the average training set error, thus demonstrating the transferability of the parameters. The root-mean-square error for gas−water, gas−hexadecane, and water−octanol transfer are 0.53, 0.38, and 0.58 log P units, respectively. Because the solvation calculation takes on average only about 0.34 s per molecule on a 700 MHz Pentium CPU and contains atom types for essentially all drug molecules, it is suitable for real-time calculations of the ADME properties of molecules in virtual ligand screening libraries.

Atomic Radii for Continuum Electrostatics Calculations on Nucleic Acids

The accuracy of continuum electrostatic models, in which the solvent is represent as a featureless dielectric medium, depends sensitively on the choice of the atomic radii used for setting the dielectric boundary between the solute and the solvent. Here, a set of optimal atomic radii for performing accurate continuum electrostatic calculations on naturally occurring nucleic acids is parametrized using molecular dynamics simulations and free energy perturbation calculations with explicit water molecules. Small model compounds constituting the building blocks of nucleic acids (base, sugar, phosphate) as well as combinations of these units (nucleoside, diphosphate sugar) are chosen to get proper representation of all the molecular moieties found in nucleic acids. Different conformations of the model compounds representative of both A-form and B-form conformations of DNA are considered. An initial set of atomic radii is first determined using a statistical mechanical analysis based on the average radial solvent charge density calculated from molecular dynamics simulations with explicit water molecules. Minor adjustments are then made to refine the radii to reproduce quantitatively the electrostatic contribution to the solvation free energy calculated by free energy perturbation techniques. The ability of the continuum dielectric approximation to accurately represent the free energy associated with base pairing is also considered by examining all possible normal and mismatched base pairs. In all cases, the agreement between molecular dynamics free energy perturbations with explicit water molecules and the continuum electrostatic calculations with optimized atomic radii is excellent. As a final illustration, the free energy profile is calculated for AT and GC base pairs as a function of base pair separation. Continuum electrostatic calculations with the optimized atomic radii provides a computationally inexpensive approach to study nucleic acids and their complexes with proteins.

The Effects of Ionic Strength on Protein Stability: The Cold Shock Protein Family

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.