Evaluating the energetics of empty cavities and internal mutations in proteins.

The energetics of cavity formation in proteins is evaluated with two different approaches and results are analyzed and compared to experimental data. In the first approach, free energy of cavity formation is extracted by RMS fitting from the distribution of numbers of cavities, N, with different volumes, Vcav, in 80 high-resolution protein structures. It is assumed that the distribution of number of cavities according to their volume follows the Boltzmann law, N(Vcav) = exp [(-a.Vcav-b)/kT], or its simplified form. Specific energy cost of cavity formation, a, extracted by RMS fitting from these distributions is compared to a values extracted from experimental free energies of cavity formation in T4 lysozyme fitted to similar expressions. It is found that fitting of both sets of data leads to similar magnitudes and uncertainties in the calculated free energy values. It is shown that Boltzmann-like distribution of cavities can be derived for a simple model of an equilibrium interconversion between mutants in an extracellular system. We, however, suggest that a partitioning into cavity-dependent and cavity-independent terms may lose meaning when one attempts to describe mutation effects on protein stability in terms of specific free energy contributions. As an alternative approach, a direct molecular mechanics evaluation is attempted of T4 lysozyme destabilization by five single cavity-creating mutations. The calculations are based on the approach used in calculations of the energetics of packing defects in crystals. For all mutations calculated destabilizations agree with the corresponding experimental values within +/-0.6 kcal/mol. A computational relaxation of the mutant was most difficult to achieve for the mutation producing the smallest cavity. However, calculations do not always reproduce crystallographically observed contraction/expansion of cavities. It is suggested that this may be related to usually observed large RMS differences (> 1 A) between crystallographic and energy-minimized protein structures, and thus correct energetics might be easier to calculate than the correct geometry.     

Hydrophilicity of cavities in proteins

Water molecules inside cavities in proteins constitute integral parts of the structure. We have sought a quantitative measure of the hydrophilicity of the cavities by calculating energies and free energies of introducing a water molecule into these cavities. A threshold value of the water-protein interaction energy at −12 kcal/mol was found to be able to distinguish hydrated from empty cavities. It follows that buried waters have entropy comparable to that of liquid water or ice. A simple consistent picture of the energetics of the buried waters provided by this study enabled us to address the reliability of buried waters assigned in experiments.     

Accessibility to internal cavities and ligand binding sites monitored by protein crystallographic thermal factors

Protein structures are flexible both in solution and in the solid state. X-ray crystallographically determined thermal factors monitor the flexibility of protein atoms. A method utilizing such factors is proposed to delineate protein regions through which a ligand can exchange between binding site and bulk solvent. It is based on the assumption that thermally excited protein regions are excellent candidates for opening a ligand channel. Computationally simple and inexpensive, the method analyzes directions from which thermal factors can propagate within the protein, resulting in thermal motion paths (TMPs). Applications to engineered T4 lysozymes, where an artificial internal cavity can host hydrophobic molecules, and to sperm whale myoglobins, where the active site is completely buried, yielded results in agreement with other independent structural observations and with previous hypotheses. Further new features could also be suggested. The proposed TMP analysis could aid molecular dynamics simulation studies as well as time-resolved and site-directed mutagenesis experimental studies, especially given its modest computational expense and its direct roots in experimental results based on thermal factors determined in high-resolution crystallographic studies. Proteins 31:201–213,1998. © 1998 Wiley-Liss, Inc.     

Statistical and molecular dynamics studies of buried waters in globular proteins

Buried solvent molecules are common in the core of globular proteins and contribute to structural stability. Folding necessitates the burial of polar backbone atoms in the protein core, whose hydrogen-bonding capacities should be satisfied on average. Whereas the residues in alpha-helices and beta-sheets form systematic main-chain hydrogen bonds, the residues in turns, coils and loops often contain polar atoms that fail to form intramolecular hydrogen bonds. The statistical analysis of 842 high resolution protein structures shows that well-resolved, internal water molecules preferentially reside near residues without alpha-helical and beta-sheet secondary structures. These buried waters most often form primary hydrogen bonds to main-chain atoms not involved in intramolecular hydrogen bonds, providing strong evidence that hydrating main-chain atoms is a key structural role of buried water molecules. Additionally, the average B-factor of protein atoms hydrogen-bonded to waters is smaller than that of protein atoms forming intramolecular hydrogen bonds, and the average B-factor of water molecules involved in primary hydrogen bonds with main-chain atoms is smaller than the average B-factor of water molecules involved in secondary hydrogen bonds to protein atoms that form concurrent intramolecular hydrogen bonds. To study the structural coupling between internal waters and buried polar atoms in detail we simulated the dynamics of wild-type FKBP12, in which a buried water, Wat137, forms one side-chain and multiple main-chain hydrogen bonds. We mutated E60, whose side-chain hydrogen bonds with Wat137, to Q, N, S or A, to modulate the multiplicity and geometry of hydrogen bonds to the water. Mutating E60 to a residue that is unable to form a hydrogen bond with Wat137 results in reorientation of the water molecule and leads to a structural readjustment of residues that are both near and distant to the water. We predict that the E60A mutation will result in a significantly reduced affinity of FKBP12 for its ligand FK506. The propensity of internal waters to hydrogen bond to buried polar atoms suggests that ordered water molecules may constitute fundamental structural components of proteins, particularly in regions where alpha-helical or beta-sheet secondary structure is not present.     

Internal cavities and buried waters in globular proteins

A fast algorithm that detects internal cavities in proteins and predicts the positions of buried water molecules is described. The cavities are characterized in terms of volume, surface area, polarity, and the presence of bound waters. The algorithm is applied to 12 proteins whose structures are known to high resolution and successfully predicts the locations of over 80% of internal water molecules. Most proteins are found to have a number of internal cavities ranging in volume from 10 to 180 A3. Some of these cavities contain water and some do not, with the probability of containing a buried water increasing with cavity size. However, many large cavities are found to be empty (i.e., they do not contain a crystallographically determined water). For multidomain proteins over half of the total cavity volume is at the interdomain interface. Possible implications for the energetics of cavity formation and for the functional role of internal cavities are discussed.     

Hydrogen-bonding classes in proteins and their contribution to the unfolding reaction

This paper proposes to assess hydrogen-bonding contributions to the protein stability, using a set of model proteins for which both X-ray structures and calorimetric unfolding data are known. Pertinent thermodynamic quantities are first estimated according to a recent model of protein energetics based on the dissolution of alkyl amides. Then it is shown that the overall free energy of hydrogen-bond formation accounts for a hydrogen-bonding propensity close to helix-forming tendencies previously found for individual amino acids. This allows us to simulate the melting curve of an alanine-rich helical 50-mer with good precision. Thereafter, hydrogen-bonding enthalpies and entropies are expressed as linear combinations of backbone-backbone, backbone-side-chain, side-chain-backbone, and side-chain-side-chain donor-acceptor contributions. On this basis, each of the four components shows a different free energy versus temperature trend. It appears that structural preference for side-chain-side-chain hydrogen bonding plays a major role in stabilizing proteins at elevated temperatures.     

Electrostatic pKa computations in proteins: role of internal cavities

The solvent accessible surface area (SASA) algorithm is conventionally used to characterize protein surfaces in electrostatic energy computations of proteins. Unfortunately, it often fails to find narrow cavities inside a protein. As a consequence pK(a) computations based on this algorithm perform badly. In this study a new cavity-algorithm is introduced, which solves this problem and provides improved pK(a) values. The procedure is applied to 20 pK(a) values of titratable groups introduced as point mutations in SNase variants, where crystal structures are available. The computations of these pK(a)s are particular challenging, since they are placed in a rather hydrophobic environment. For nine mutants, where the titratable residue is in contact with a large cavity, the RMSD(pKa) between computed and measured pK(a) values is 2.04, which is a considerable improvement as compared to the original results obtained with Karlsberg(+) (http://agknapp.chemie.fu-berlin.de/karlsberg/) that yielded an RMSD(pKa) of 8.8. However, for 11 titratable residues the agreement with experiments remains poor (RMSD(pKa) = 6.01). Considering 15 pK(a)s of SNase, which are in a more conventional less hydrophobic protein environment, the RMSD(pKa) is 2.1 using the SASA-algorithm and 1.7 using the new cavity-algorithm. The agreement is reasonable but less good than what one would expect from the general performance of Karlsberg(+) indicating that SNase belongs to the more difficult proteins with respect to pK(a) computations. We discuss the possible reasons for the remaining discrepancies between computed and measured pK(a)s.     

Thermal stability determinants of chicken egg-white lysozyme core mutants: hydrophobicity, packing volume, and conserved buried water molecules.

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal denaturation transition (Tm) values of these core constructs range from 60.9 to 77.3 degrees C, extending an earlier, more limited investigation on thermostability. The Tm values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9 A) and I55L/S91T/D101S (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of Tm for the S91D mutant was compared with that of WT enzyme. The pKa of S91D is 1.2 units higher in the native than in the denatured state, corresponding to delta delta G298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.     

Slow internal dynamics of membrane proteins in mechanisms of protease-induced aggregation of platelets

Room temperature tryptophan phosphorescence (RTTP) of suspensions of human platelets was studied. RTTP spectra and decay kinetics of both intact platelets and those after short-term incubation with low concentrations of thrombin or trypsin (0.3-50 microg/ml) were investigated. Protease-induced changes in the RTTP lifetime of platelets were observed, and interpreted in terms of the slow internal dynamics of membrane protein modification. The functional role of membrane protein internal dynamics is discussed in the context of platelet aggregation and signal transduction processes.     

Conserved internal hydration motifs in protein kinases

Crystal structures of diverse protein kinase catalytic subunits reveal a number of water molecules whose positions within the protein core are better preserved than amino acid types in many functionally important locations. It remains unknown whether they play any particular role, and whether their removal, disturbing local interaction patterns to no smaller degree than amino acid mutations, can affect kinase stability and function. In this study, we apply an array of computational approaches to characterize hydration of kinase catalytic subunits. First, we systematically screen multiple crystal structures with the use of a simplified hydration model in order to determine the distribution of internal solvent and the degree of its conservation. Second, we analyze water structure, dynamics and binding affinity to buried hydration sites in a number of kinases, also taking into account their variable functional state. We find that a large portion of buried solvent is dynamic, possibly contributing to kinase conformational changes related to the activation process. In turn, binding free energies of some of tightly bound conserved water molecules to different kinases tend to shift in a similar manner following the change of their functional state. This finding highlights the likely specific role of internal solvent in fine tuning local protein plasticity.     

Crystal structures of subtilisin BPN' variants containing disulfide bonds and cavities: concerted structural rearrangements induced by mutagenesis

The X-ray structures of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26-Cys232 and Cys29-Cys119, induced larger changes than did Cys24-Cys87 and Cys22-Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as alpha-helices and beta-sheets indicated systematic long-range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that border the cavities.     

Cavities and packing at protein interfaces.

An analysis of internal packing defects or "cavities" (both empty and water-containing) within protein structures has been undertaken and includes 3 cavity classes: within domains, between domains, and between protein subunits. We confirm several basic features common to all cavity types but also find a number of new characteristics, including those that distinguish the classes. The total cavity volume remains only a small fraction of the total protein volume and yet increases with protein size. Water-filled "cavities" possess a more polar surface and are typically larger. Their constituent waters are necessary to satisfy the local hydrogen bonding potential. Cavity-surrounding atoms are observed to be, on average, less flexible than their environments. Intersubunit and interdomain cavities are on average larger than the intradomain cavities, occupy a larger fraction of their resident surfaces, and are more frequently water-filled. We observe increased cavity volume at domain-domain interfaces involved with shear type domain motions. The significance of interfacial cavities upon subunit and domain shape complementarity and the protein docking problem, as well as in their structural and functional role in oligomeric proteins, will be discussed. The results concerning cavity size, polarity, solvation, general abundance, and residue type constituency should provide useful guidelines for protein modeling and design.     

Dental caries in wild primates: Interproximal cavities on anterior teeth

Dental caries has been reported in a variety of primates, although it is still considered rare in wild populations. In this study, 11 catarrhine primate taxa (n = 339 individuals; 7946 teeth) were studied for the presence of caries. A differential diagnosis of lesions in interproximal regions of anterior teeth was undertaken, since they had been previously described as both carious and non-carious in origin. Each permanent tooth was examined macroscopically, with severity and position of lesions recorded. Two specimens were examined further, using micro-CT scans to assess demineralization. Differential diagnosis confirmed the cariogenic nature of interproximal cavities on anterior teeth (ICATs). Overall results show 3.3% of all teeth (i.e., anterior and posterior teeth combined) were carious (n = 262), with prevalence varying among species from 0% to >7% of teeth affected. Those with the highest prevalence of ICATs include Pan troglodytes verus (9.8% of anterior teeth), Gorilla gorilla gorilla (2.6%), Cercopithecus denti (22.4%), Presbytis femoralis (19.5%), and Cercopithecus mitis (18.3%). ICATs make up 87.9% of carious lesions on anterior teeth. These results likely reflect dietary and food processing differences among species, but also between the sexes (e.g., 9.3% of all female P. troglodytes verus teeth were carious vs. 1.8% in males). Processing cariogenic fruits and seeds with the anterior dentition (e.g., wadging) likely contributes to ICAT formation. Further research is needed in living primate populations to ascertain behavioral/dietary influences on caries occurrence. Given the presence of ICATs in frugivorous primates, their diagnosis in archaeological and paleontological specimens may shed light on diet and food processing behaviors in fossil primates.     

Arginyl residues and thermal stability in proteins

Guanidination and amidination of bovine serum albumin, yeast enolase and yeast alcohol dehydrogenase were accompanied by increases in thermal stability at lower extents of modification. Decreases in thermal stability result from greater modification. These results support suggestions that surface guanidino groups (arginyl groups) are an important factor in thermal stability of proteins.     

Some analyses of aquatic plants and their waters

Values are given of chloride, sulphate, phosphate, calcium, sodium, potassium and total nitrogen on aquatic plant material, both water and acid washed, and the corresponding waters.Resumé Ci inclus les resultats de l'analyses de plantes aquatiques sont donnés dont les constituents sont du chlore, du sulfate, du phosphate, du calcium, du potassium, du sodium et du nitrogène.     

A "structural" water molecule in the family of fatty acid binding proteins

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1')anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 A2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 +/- 1.8 A3 and 24.7 +/- 2.2 A3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in beta-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.     

Role of flexibility and polarity as determinants of the hydration of internal cavities and pockets in proteins

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized. This might explain differences in the patterns of hydration observed in crystal structures obtained at cryogenic and room temperature conditions. The ability of molecular dynamics simulations to determine the most likely sites of water binding in internal pockets and cavities depends on its efficiency in sampling the hydration of internal sites and alternative protein and water conformations. This can be enhanced significantly by performing multiple molecular dynamics simulations as well as simulations started from different initial hydration states.     

Conserved water molecules in the specificity pocket of serine proteases and the molecular mechanism of Na+ binding

Conservation of clusters of buried water molecules is a structural motif present throughout the serine protease family. Frequently, these clusters are shaped as water channels forming extensive hydrogen-bonding networks linked to the protein backbone. The most conspicuous example is the water channel present in the specificity pocket of trypsin and thrombin. In thrombin, other vitamin K-dependent proteases, and some complement factors, Na⁺ binds in this water channel and enhances allosterically the catalytic activity of the enzyme, whereas digestive and fibrinolytic proteases are devoid of such regulation. A comparative analysis of proteases with and without Na⁺ binding capability reveals the role of the water channel in maintaining the structural organization of the specificity pocket and in Na⁺ coordination. This enables the formulation of a molecular mechanism for Na⁺ binding in thrombin and leads to the identification of the structural changes necessary to engineer a functional Na⁺ site and enhanced catalytic activity in trypsin and other proteases. Proteins 30:34–42, 1998. © 1998 Wiley-Liss, Inc.     

Internal Water Molecules as Mobile Polar Groups for Light-Induced Proton Translocation in Bacteriorhodopsin and Rhodopsin as Studied by Difference FTIR Spectroscopy

FTIR spectroscopy is advantageous for detecting changes in polar chemical bonds that participate in bacteriorhodopsin function. Changes in H-bonding of Asp85, Asp96, the Schiff base, and internal water molecules around these residues upon the formation of the L, M, and N photo-intermediates of bacteriorhodopsin were investigated by difference FTIR spectroscopy. The locations and the interactions of these water molecules with the amino acid residues were further revealed by use of mutant pigments. The internal water molecules in the cytoplasmic domain probably work as mobile polar groups in an otherwise apolar environment and act to stabilize the L intermediate, and carrying a proton between the Schiff base and the proton acceptor or donor. Similar internal water molecules were shown to be present in bovine rhodopsin.