SIMPLE estimate of the free energy change due to aliphatic mutations: superior predictions based on first principles

The bioinformatics revolution of the last decade has been instrumental in the development of empirical potentials to quantitatively estimate protein interactions for modeling and design. Although computationally efficient, these potentials hide most of the relevant thermodynamics in 5-to-40 parameters that are fitted against a large experimental database. Here, we revisit this longstanding problem and show that a careful consideration of the change in hydrophobicity, electrostatics, and configurational entropy between the folded and unfolded state of aliphatic point mutations predicts 20-30% less false positives and yields more accurate predictions than any published empirical energy function. This significant improvement is achieved with essentially no free parameters, validating past theoretical and experimental efforts to understand the thermodynamics of protein folding. Our first principle analysis strongly suggests that both the solute-solute van der Waals interactions in the folded state and the electrostatics free energy change of exposed aliphatic mutations are almost completely compensated by similar interactions operating in the unfolded ensemble. Not surprisingly, the problem of properly accounting for the solvent contribution to the free energy of polar and charged group mutations, as well as of mutations that disrupt the protein backbone remains open.     

Filling small, empty protein cavities: structural and energetic consequences

Most proteins contain small cavities that can be filled by replacing cavity-lining residues by larger ones. Since shortening mutations in hydrophobic cores tend to destabilize proteins, it is expected that cavity-filling mutations may conversely increase protein stability. We have filled three small cavities in apoflavodoxin and determined by NMR and equilibrium unfolding analysis their impact in protein structure and stability. The smallest cavity (14 A3) has been filled, at two different positions, with a variety of residues and, in all cases, the mutant proteins are locally unfolded, their structure and energetics resembling those of an equilibrium intermediate of the thermal unfolding of the wild-type protein. In contrast, two slightly larger cavities of 20 A3 and 21 A3 have been filled with Val to Ile or Val to Leu mutations and the mutants preserve both the native fold and the equilibrium unfolding mechanism. From the known relationship, observed in shortening mutations, between stability changes and the differential hydrophobicity of the exchanged residues and the volume of the cavities, the filling of these apoflavodoxin cavities is expected to stabilize the protein by approximately 1.5 kcal mol(-1). However, both urea and thermal denaturation analysis reveal much more modest stabilizations, ranging from 0.0 kcal mol(-1) to 0.6 kcal mol(-1), which reflects that the accommodation of single extra methyl groups in small cavities requires some rearrangement, necessarily destabilizing, that lowers the expected theoretical stabilization. As the size of these cavities is representative of that of the typical small, empty cavities found in most proteins, it seems unlikely that filling this type of cavities will give rise to large stabilizations.     

Energetics of cell-cell and cell-biopolymer interactions

The energy vs distance balance of cell suspensions (in the presence and in the absence of extracellular biopolymer solutions) is studied, not only in the light of the classical Derjaguin-Landau-Verwey-Over-beek (DLVO) theory (which considered just the electrostatic (EL) and Lifshitz-van der Waals (LW) interactions), but also by taking electron-acceptor/electron-donor, or Lewis acid-base (AB) and osmotic (OS) interactions into account. Since cell surfaces, as well as many biopolymers tend to have strong monopolar electron-donor properties, they are able to engage in a strong mutual AB repulsion when immersed in a polar liquid such as water. The effects of that repulsion have been observed earlier in the guise of hydration pressure. The AB repulsion is, at close range, typically one or two orders of magnitude stronger than the EL repulsion, but its rate of decay is much steeper. In most cases, AB interactions are quantitatively the dominant factor in cell stability (when repulsive) and in “hydrophobic interactions” (when attractive). OS interactions exerted by extracellularly dissolved biopolymers are weak, but their rate of decay is very gradual, so OS repulsions engendered by biopolymer solutions may be of importance in certain long-range interactions. OS interactions exerted by biopolymers attached to cells or particles (e.g., by glycocalix glycoproteins), are very short-ranged and usually are negligibly small in comparison with the other interaction forces, in aqueous media.     

Side-chain contributions to membrane protein structure and stability

The molecular forces that stabilize membrane protein structure are poorly understood. To investigate these forces we introduced alanine substitutions at 24 positions in the B helix of bacteriorhodopsin and examined their effects on structure and stability. Although most of the results can be rationalized in terms of the folded structure, there are a number of surprises. (1) We find a remarkably high frequency of stabilizing mutations (17%), indicating that membrane proteins are not highly optimized for stability. (2) Helix B is kinked, with the kink centered around Pro50. The P50A mutation has no effect on stability, however, and a crystal structure reveals that the helix remains bent, indicating that tertiary contacts dominate in the distortion of this helix. (3) We find that the protein is stabilized by about 1kcal/mol for every 38A(2) of surface area buried, which is quite similar to soluble proteins in spite of their dramatically different environments. (4) We find little energetic difference, on average, in the burial of apolar surface or polar surface area, implying that van der Waals packing is the dominant force that drives membrane protein folding.     

The dominant role of side-chain backbone interactions in structural realization of amino acid code. ChiRotor: a side-chain prediction algorithm based on side-chain backbone interactions

The basic differences between the 20 natural amino acid residues are due to differences in their side-chain structures. This characteristic design of protein building blocks implies that side-chain-side-chain interactions play an important, even dominant role in 3D-structural realization of amino acid codes. Here we present the results of a comparative analysis of the contributions of side-chain-side-chain (s-s) and side-chain-backbone (s-b) interactions to the stabilization of folded protein structures within the framework of the CHARMm molecular data model. Contrary to intuition, our results suggest that side-chain-backbone interactions play the major role in side-chain packing, in stabilizing the folded structures, and in differentiating the folded structures from the unfolded or misfolded structures, while the interactions between side chains have a secondary effect. An additional analysis of electrostatic energies suggests that combinatorial dominance of the interactions between opposite charges makes the electrostatic interactions act as an unspecific folding force that stabilizes not only native structure, but also compact random conformations. This observation is in agreement with experimental findings that, in the denatured state, the charge-charge interactions stabilize more compact conformations. Taking advantage of the dominant role of side-chain-backbone interactions in side-chain packing to reduce the combinatorial problem, we developed a new algorithm, ChiRotor, for rapid prediction of side-chain conformations. We present the results of a validation study of the method based on a set of high resolution X-ray structures.     

Impacts of hydrophobicity and ionicity of phendione-based cobalt(II)/(III) complexes on binding with bovine serum albumin

Abstract

For efficient designing of metallodrugs, it is imperative to analyse the binding affinity of those drugs with drug-carrying serum albumins to comprehend their structure–activity correlation for biomedical applications. Here, cobalt(II) and cobalt(III) complexes comprising three phendione ligands, [Co(phendione)₃]Cl₂ (1) and [Co(phendione)₃]Cl₃ (2), where, phendione = 1,10-phenanthroline-5,6-dione, has been chosen to contrast the impact of their hydrophobicity and ionicity on binding with bovine serum albumin (BSA) through spectrophotometric titrations. The attained hydrophobicity values using octanol/water partition coefficient method manifested that complex 1 is more hydrophobic than complex 2, which could be attributed to lesser charge on its coordination sphere. The interaction of complexes 1 and 2 with BSA using steady state fluorescence studies revealed that these complexes quench the intrinsic fluorescence of BSA through static mechanism, and the extent of quenching and binding parameters are higher for complex 2. Further thermodynamics of BSA-binding studies revealed that complexes 1 and 2 interact with BSA through hydrophobic and hydrogen bonding/van der Waals interactions, respectively. Further, UV–visible absorption, circular dichroism and synchronous fluorescence studies confirmed the occurrence of conformational and microenvironmental changes in BSA upon binding with complexes 1 and 2. Molecular docking studies have also shown that complex 2 has a higher binding affinity towards BSA as compared to complex 1. This sort of modification of ionicity and hydrophobicity of metal complexes for getting desirable binding mode/strength with drug transporting serum albumins will be a promising pathway for designing active and new kind of metallodrugs for various biomedical applications.

Communicated by Ramaswamy H. Sarma     

Determination of the second virial coefficient of bovine serum albumin under varying pH and ionic strength by composition-gradient multi-angle static light scattering

Composition-gradient multi-angle static light scattering (CG-MALS) is an emerging technique for the determination of intermolecular interactions via the second virial coefficient B₂₂. With CG-MALS, detailed studies of the second virial coefficient can be carried out more accurately and effectively than with traditional methods. In addition, automated mixing, delivery and measurement enable high speed, continuous, fluctuation-free sample delivery and accurate results. Using CG-MALS we measure the second virial coefficient of bovine serum albumin (BSA) in aqueous solutions at various values of pH and ionic strength of a univalent salt (NaCl). The systematic variation of the second virial coefficient as a function of pH and NaCl strength reveals the net charge change and the isoelectric point of BSA under different solution conditions. The magnitude of the second virial coefficient decreases to 1.13 x 10⁻⁵ ml*mol/g² near the isoelectric point of pH 4.6 and 25 mM NaCl. These results illuminate the role of fundamental long-range electrostatic and van der Waals forces in protein-protein interactions, specifically their dependence on pH and ionic strength. Electronic supplementary material The online version of this article (doi:10.1007/s10867-014-9367-7) contains supplementary material, which is available to authorized users.     

Effect of T-R conformational change on sickle-cell hemoglobin interactions and aggregation

We compare the role of a conformational switch and that of a point mutation in the thermodynamic stability of a protein solution and in the consequent propensity toward aggregation. We study sickle-cell hemoglobin (HbS), the beta6 Glu-Val point mutant of adult human hemoglobin (HbA), in its R (CO-liganded) conformation, and compare its aggregation properties to those of both HbS and HbA in their T (unliganded) conformation. Static and dynamic light scattering measurements performed for various hemoglobin concentrations showed critical divergences with mean field exponents as temperature was increased. This allowed determining spinodal data points T(S)(c) by extrapolation. These points were fitted to theoretical expressions of the T(S)(c) spinodal line, which delimits the region where the homogeneous solution becomes thermodynamically unstable against demixing in two sets of denser and dilute mesoscopic domains, while remaining still liquid. Fitting provided model-free numerical values of enthalpy and entropy parameters measuring the stability of solutions against demixing, namely, 93.2 kJ/mol and 314 J/ degrees K-mol, respectively. Aggregation was observed also for R-HbS, but in amorphous form and above physiological temperatures close to the spinodal, consistent with the role played in nucleation by anomalous fluctuations governed by the parameter epsilon = (T - T(S))/T(S). Fourier transform infrared (FTIR) and optical spectroscopy showed that aggregation is neither preceded nor followed by denaturation. Transient multiple interprotein contacts occur in the denser liquid domains for R-HbS, T-HbS, and T-HbA. The distinct effects of their specific nature and configurations, and those of desolvation on the demixing and aggregation thermodynamics, and on the aggregate structure are highlighted.     

Atomic contacts in protein structures. A detailed analysis of atomic radii, packing, and overlaps

A rigorous quantitative assessment of atomic contacts and packing in native protein structures is presented. The analysis is based on optimized atomic radii derived from a set of high-resolution protein structures and reveals that the distribution of atomic contacts and overlaps is a structural constraint in proteins, irrespective of structural or functional classification and size. Furthermore, a newly developed method for calculating packing properties is introduced and applied to sets of protein structures at different levels of resolution. The results show that limited resolution yields decreasing packing quality, which underscores the relevance of packing considerations for structure prediction, design, dynamics, and docking.     

Orientation of the water molecules of hydration of human serum albumin

Through contact-angle measurements with a number of liquids, on layers of hydrated human serum albumin (HSA), built on anisotropic ultrafilter membranes, the apolar, Lifshitz-van der Waals surface tension component, as well as the polar, electron-acceptor and electron-donor parameters of the hydrated layers could be determined. From these data, it was found that the degree of orientation of the water molecules of hydration of HSA is 98% in the first layer of hydration and 30% of the second layer. The water molecules of hydration are oriented with the H atoms closest to, and the O atoms farthest from, the protein surface.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.     

Antifreeze protein from shorthorn sculpin: Identification of the ice-binding surface

Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine-rich, alpha-helical antifreeze proteins (AFPs). The major serum isoform (SS-8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic alpha-helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the [11-20] plane of ice in the <-1102> direction. To test this hypothesis, a series of SS-8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant alpha-helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala-rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.     

Highly accurate method for ligand-binding site prediction in unbound state (apo) protein structures

This article describes a new method for predicting ligand-binding sites of proteins. The method involves calculating the van der Waals interaction energy between a protein and probes placed on the protein surface, and then clustering the probes with attractive interaction to find the energetically most favorable locus. In 80% (28/35) of the test cases, the ligand-binding site was successfully predicted on a ligand-bound protein structure, and in 77% (27/35) was successfully predicted on an unbound structure. Our method was used to successfully predict ligand-binding sites unaffected by induced-fit as long as its scales were not very large, and it contributed to a significant improvement in prediction with unbound state protein structures. This represents a significant advance over conventional methods in detecting ligand-binding sites on uncharacterized proteins. Moreover, our method can predict ligand-binding sites with a narrower locus than those achieved using conventional methods.     

Fractal nature of protein surface roughness: a note on quantification of change of surface roughness in active sites,before and after binding

Year 2010 marked the 25th year since we came to know that roughness of a protein surface has fractal symmetry. Ever since the publication of Lewis and Rees' paper, hundreds of works from a spectrum of perspectives have established that fractal dimension (FD) can be considered as a reliable marker that describes roughness of protein surface objectively. In this article, we introduce readers to the fundamentals of fractals and present categorical biophysical and geometrical reasons as to why FD‐based constructs can describe protein surface roughness more accurately. We then review the commonality (and the lack of it) between numerous approaches that have attempted to investigate protein surface with fractal measures, before exploring the patterns in the results that they have produced. Apart from presenting the genealogy of approaches and results, we present an analysis that quantifies the difference in surface roughness in stretches of protein surface containing the active site, before and after binding to ligands, to underline the utility of FD‐based measures further. It has been found that surface stretches containing the active site, in general, undergo a significant increment in its roughness after binding. After presenting the entire repertoire of FD‐based surface roughness studies, we talk about two yet‐unexplored problems where application of FD‐based techniques can help in deciphering underlying patterns of surface interactions. Finally, we list the limitations of FD‐based constructs and put down several precautions that one must take while working with them. Copyright © 2013 John Wiley & Sons, Ltd.     

Computational redesign of the SHV-1 beta-lactamase/beta-lactamase inhibitor protein interface

β-lactamases are enzymes that catalyze the hydrolysis of β-lactam antibiotics. β-lactamase/β-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. BLIP is a 165 amino acid protein that inhibits several class A β-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 β-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 Å resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.     

Surface thermodynamic properties and chromatographic and salting-out behavior of IgA and other serum proteins

By means of contact angle determinations with two liquids, on hydrated as well as on dried protein layers, the long-range and the short-range contributions to the protein surface tensions, and from these the protein (G ₁₃₁) and the protein-ligand (G ₁₃₂) free energies of interaction in aqueous media, were determined. For human serum albumin (HSA), human IgG, and human IgA, the differences between G ₁₃₁ ^HYDRATED and G ₁₃₁ ^DRY were connected with the behavior of these proteins in low concentrations of (NH₄)₂SO₄ versus saturated (NH₄)₂SO₄ solutions. By interpolation, intermediate points are found that correlate well with the known salting-out properties of these three proteins. On the basis of the data, it is predicted that the precipitation of IgG by 1/3 saturated (NH₄)₂SO₄ is preventable, or reversible, by the admixture of 15% dimethylsulfoxide; both predictions are confirmed experimentally. From the G ₁₃₂ values found, it is shown that HSA and IgG should attach to phenyl ligands under physiological conditions, but that IgA is so hydrophilic that it only can adhere to phenyl ligands after partial dehydration brought about when admixed with 1 M (NH₄)₂SO₄. Closer analysis of the values obtained for the long-range and short-range components of the surface tensions of HSA, IgG, and IgA allow deeper insight into their functional, chemical, and physicochemical properties.     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998