Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.     

Energetics of protein homodimerization: effects of water sequestering on the formation of beta-lactoglobulin dimer

Transient protein-protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein-protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine beta lg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. DeltaH and deltaC(p) were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near-UV CD spectra proved to be independent of protein concentration, indicating a rigid body-like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer's 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental deltaC(p). Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces.     

Interaction of C-phycocyanin with lipid monolayers under nitrogen and in the presence of air

The surface interaction of C-phycocyanin with lipids was studied using the monolayer technique. The surface activity of the protein was found to be higher at the lipid-water interface than at the nitrogen-water interface, particularly at high surface pressures of the lipid monolayer. The maximum initial surface pressures beyond which phycocyanin could not penetrate the dipalmitoylphosphatidylcholine and monogalactosyldiglycerol monolayers were 27 and 30 mN m-1, respectively. Below these values the protein demonstrated preferential interaction with the monogalactosyldiglycerol monolayer. The surface properties of the unfolded protein at pH 2.5 at the lipid-water interface were compared with those of the protein at pH 7.0. Higher affinity of the three-dimensional structure of the protein to lipid monolayers was observed, in particular by high subphase protein concentration. When the lipid films were subjected to oxidation stress by exposure to air, the surface properties of C-phycocyanin and dipalmitoylphosphatidylcholine were not greatly affected but the surface activity of monogalactosyldiacylglycerol was reduced dramatically by autoxidation. The oxidation of monogalactosyldiacylglycerol could not be prevented by the introduction of C-phycocyanin molecules at the lipid-water interface.     

The role of water coordination in the pH-dependent gating of hAQP10

Human aquaporin 10 (hAQP10) is an aquaglyceroporin that assists in maintaining glycerol flux in adipocytes during lipolysis at low pH. Hence, a molecular understanding of the pH-sensitive glycerol conductance may open up for drug development in obesity and metabolically related disorders. Control of hAQP10-mediated glycerol flux has been linked to the cytoplasmic end of the channel, where a unique loop is regulated by the protonation status of histidine 80 (H80). Here, we performed unbiased molecular dynamics simulations of three protonation states of H80 to unravel channel gating. Strikingly, at neutral pH, we identified a water coordination pattern with an inverted orientation of the water molecules in vicinity of the loop. Protonation of H80 results in a more hydrophobic loop conformation, causing loss of water coordination and leaving the pore often dehydrated. Our results indicate that the loss of such water interaction network may be integral for the destabilization of the loop in the closed configuration at low pH. Additionally, a residue unique to hAQP10 (F85) reveals structural importance by flipping into the channel in correlation with loop movements, indicating a loop-stabilizing role in the closed configuration. Taken together, our simulations suggest a unique gating mechanism combining complex interaction networks between water molecules and protein residues at the loop interface. Considering the role of hAQP10 in adipocytes, the detailed molecular insights of pH-regulation presented here will help to understand glycerol pathways in these cells and may assist in drug discovery for better management of human adiposity and obesity.     

The refinement and the structure of the dimer of alpha-chymotrypsin at 1.67-A resolution

The two molecules of the asymmetric unit of the pH 3.5 conformer of alpha-chymotrypsin have been refined at 1.67-A resolution using restrained least squares methods with Hendrickson's program (PROLSQ). The final R factor is 0.179 (including 247 water molecules). The folding of the main chain of the independent molecules is the same within experimental error but the same does not generally apply to the side chain stereochemistry. From this we conclude that the folding of a protein structure is basically independent of most of the detailed stereochemistry of its side chains. The side chains of the interface region between the independent molecules display pronounced asymmetry. This asymmetry suggests that dynamic and asymmetrical structural changes take place at the time of oligomerization leading to more energetically favorable interactions for the dimer. Comparison of the structures of the independent molecules of alpha-chymotrypsin with the structure of monomeric gamma-chymotrypsin revealed that although the folding of the three molecules is essentially the same, numerous and significant differences pervade the side chain stereochemistry attributable to general flexibility. The specificity site of alpha-chymotrypsin is occupied by ordered water molecules in a similar way to gamma-chymotrypsin and other proteins. Some of these water molecules are displaced when substrate binds to the enzyme, while the others appear to help identify and position the aromatic side chain in catalysis.     

A "solvated rotamer" approach to modeling water-mediated hydrogen bonds at protein-protein interfaces

Water-mediated hydrogen bonds play critical roles at protein-protein and protein-nucleic acid interfaces, and the interactions formed by discrete water molecules cannot be captured using continuum solvent models. We describe a simple model for the energetics of water-mediated hydrogen bonds, and show that, together with knowledge of the positions of buried water molecules observed in X-ray crystal structures, the model improves the prediction of free-energy changes upon mutation at protein-protein interfaces, and the recovery of native amino acid sequences in protein interface design calculations. We then describe a "solvated rotamer" approach to efficiently predict the positions of water molecules, at protein-protein interfaces and in monomeric proteins, that is compatible with widely used rotamer-based side-chain packing and protein design algorithms. Finally, we examine the extent to which the predicted water molecules can be used to improve prediction of amino acid identities and protein-protein interface stability, and discuss avenues for overcoming current limitations of the approach.     

Conservation of water molecules in an antibody–antigen interaction

The solvation of the antibody–antigen Fv D1.3–lysozyme complex is investigated through a study of the conservation of water molecules in crystal structures of the wild-type Fv fragment of antibody D1.3, 5 free lysozyme, the wild-type Fv D1.3–lysozyme complex, 5 Fv D1.3 mutants complexed with lysozyme and the crystal structure of an idiotope (Fv D1.3)-abti-idiotope (Fv E5.2) complex. In all, there are 99 water molecules common to the wild-type and mutant antibody–lysozyme complexes. The antibody–lysozyme interface includes 25 well-ordered solvent molecules, conserved among the wild-type and mutant Fv D1.3–lysozyme complexes, which are bound directly or through other water molecules to both antibody and antigen. In addition to contributing hydrogen bonds to the antibody–antigen interaction the solvent molecules fill many interface cavities. Comparison with x-ray crystal structures of free Fv D1.3 and free lysozyme shows that 20 of these conserved interface waters in the complex were bound to one of the free proteins. Uo to 23 additional water molecules are also found in the antibody–antigen interface, however these waters do no bridge antibody and antigen and their temperature factors are much higher than those of the 25 well-ordered waters. Fifteen water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and 5 water molecules are added at the antibody–antigen interface with the formation of the complex. While the current crystal models of the D1.3–lysozyme complex do not demonstrate the increase in bound waters found in a physico-chemical study of the interaction at decreased water activities, the 25 well-ordered interface water contribute a net gain of 10 hydrogen bonds to complex stability.     

Hydrogen-tritium exchange kinetics of soybean trypsin inhibitor (Kunitz). Solvent accessibility in the folded conformation.

The hydrogen exchange kinetics of Kunitz soybean trypsin inhibitor (STI) has been studied at pH 2, 3, and 6.5. From the temperature dependence of proton exchange at low pH, THE CONTRIBUTION OF MAJOR, REVERSIBLE PROTEIN UNFOLDING To the hydrogen exchange kinetics has been determined. Exchange directly from the folded conformation is characterized by an apparent activation energy (E*app) of approximately 25 kcal/mol, close to that of the chemical exchange step. At pH 6.5 the protein is more temperature stable than at low pH, and exchange of all but congruent to 8 protons can be observed to exchange with E*app congruent to 27 kcal/mol. This implies that all but congruent to 8 protons are accessible to exchange with solvent in the solution structure of folded STI. Estimates can be made of the average number of water molecules per molecule of STI consistent with a solvent accessibility model of hydrogen exchange kinetics. These estimates indicate that very few water molecules within the protein matrix are necessary to explain the exchange data. Calculations are done for the STI hydrogen exchange kinetics at pH 3, 30 degrees, approximating STI structure by a sphere of radius = 18 A. These calculations indicate an average of congruent to 4 water molecules in the shell from 13 to 16 A. from the center of the molecule, while less than 1 water molecule is indicated in the innermost 13 A. These calculations also suggest that there are congruent to 190 water molecules associated with the outermost 1.5-2 A of the sphere. While these values are consistent with a hydrophobic region in the central protein matrix, they indicate more solvent accessibility in the outer 1/3 of the molecule than the static accessibility estimates made from X-ray coordinates. Our results suggest that any protein movements or fluctuations responsible for solvent accessibility in proton exchange processes are localized in the outer regions of the globular structure.     

Renaturation and interaction of ribonuclease A with AOT surfactant in reverse micelles

This study extended works on effects of solute on the percolation of reverse micelles to the effects of interactions between protein and surfactants on protein refolding by reverse micelles. The changes in percolation behavior were identified and attributed to the position of solutes in the core aqueous phase and the interaction between the solute and the surfactants. The percolation behavior of reverse micelles with solutes was related to protein renaturation and the reverse micelle. This study aims to highlight the involvement of the interface and the interaction of the protein with the surfactant during protein refolding. Ribonuclease A and AOT reverse micelles together constitute a model system considered here. The systemic parameters of the reverse micelle, water content (W(o)) and pH value, were applied to modify the interaction between the denatured protein molecules and the surfactant interface. The interactions and the locations of the protein molecules were determined from changes in percolation temperature measured by conductivity. The percolation and protein activity show that a stronger interaction of the protein molecules with surfactant corresponds to superior recovery of protein activity. The investigation concludes that the refolding of protein by reverse micelles is not only facilitated by the isolation of reverse micelles but also by the interaction due to the interface of the reverse micelle.     

X-ray and thermodynamic studies of staphylococcal nuclease variants I92E and I92K: insights into polarity of the protein interior

We have used crystallography and thermodynamic analysis to study nuclease variants I92E and I92K, in which an ionizable side-chain is placed in the hydrophobic core of nuclease. We find that the energetic cost of burying ionizable groups is rather modest. The X-ray determinations show water molecules solvating the buried glutamic acid under cryo conditions, but not at room temperature. The lysine side-chain does not appear solvated in either case. Guanidine hydrochloride (GnHCl) denaturation of I92E and I92K, done as a function of pH and monitored by tryptophan fluorescence, showed that I92E and I92K are folded in the pH range pH 3.5-9.0 and pH 5.5-9.5, respectively. The stability of the parental protein is independent of pH over a broad range. In contrast, the stabilities of I92E and I92K exhibit a pH dependence, which is quantitatively explained by thermodynamic analysis: the PK(a) value of the buried K92 is 5.6, while that of the buried E92 is 8.65. The free energy difference between burying the uncharged and charged forms of the groups is modest, about 6 kcal/mol. We also found that epsilon(app) for I92K and I92E is in the range approximately 10-12, instead of 2-4 commonly used to represent the protein interior. Side-chains 92E and 92K were uncharged under the conditions of the X-ray experiment. Both are buried completely inside the well-defined hydrophobic core of the variant proteins without forming salt-bridges or hydrogen bonds to other functional groups of the proteins. Under cryo conditions 92E shows a chain of four water molecules, which hydrate one oxygen atom of the carboxyl group of the glutamic acid. Two other water molecules, which are present in the wild-type at all temperatures, are also connected to the water ring observed inside the hydrophobic core. The ready burial of water with an uncharged E92 raises the possibility that solvent excursions into the interior also take place in the wild-type protein, but in a random, dynamic way not detectable by crystallography. Such transient excursions could increase the average polarity, and thus epsilon(app), of the protein interior.     

Antifreeze proteins at the ice/water interface: three calculated discriminating properties for orientation of type I proteins

Antifreeze proteins (AFPs) protect many plants and organisms from freezing in low temperatures. Of the different AFPs, the most studied AFP Type I from winter flounder is used in the current computational studies to gain molecular insight into its adsorption at the ice/water interface. Employing molecular dynamics simulations, we calculate the free energy difference between the hydrophilic and hydrophobic faces of the protein interacting with ice. Furthermore, we identify three properties of Type I "antifreeze" proteins that discriminate among these two orientations of the protein at the ice/water interface. The three properties are: the "surface area" of the protein; a measure of the interaction of the protein with neighboring water molecules as determined by the number of hydrogen bond count, for example; and the side-chain orientation angles of the threonine residues. All three discriminants are consistent with our free energy results, which clearly show that the hydrophilic protein face orientations toward the ice/water interface, as hypothesized from experimental and ice/vacuum simulations, are incorrect and support the hypothesis that the hydrophobic face is oriented toward the ice/water interface. The adsorption free energy is calculated to be 2-3 kJ/mol.     

Acidic groups docked to well defined wetted pockets at the core of the binding interface: a tale of scoring and missing protein interactions in CAPRI

Some challenging targets in CAPRI (T24/25 and T26) involve binding solvent accessible acidic residues at the core of the binding interface, where they are always found immersed in crystal waters. In fact, Asp and Glu residues are more likely to form part of the hydrogen bond network of their surrounding crystal water molecules than to form a buried salt bridge. Interestingly, many of the crystal waters mediating the intermolecular interactions of the acidic groups are already present in the unbound structure, reinforcing the notion that some water molecules behave as an extension of the protein structure. This is in contrast to acidic groups found in the periphery of the binding interface that form ubiquitous salt bridges that cement the high affinity complex, while at the same time they are exposed to rapidly exchanging water molecules. Because of this, dichotomy implicit solvent scoring functions fail to properly rank these complexes by prioritizing salt bridges rather than water mediated contacts. A detailed analysis of Target 24, for which our group predicted two out of the four successful homology model complex structures, and Target 26 reveal how crystal waters shape the binding cavities of acidic groups prior to binding, in agreement with the theory of anchor residues as mediators of protein recognition.     

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Interaction between bound water molecules and local protein structures: A statistical analysis of the hydrogen bond structures around bound water molecules

Water molecules play an important role in protein folding and protein interactions through their structural association with proteins. Examples of such structural association can be found in protein crystal structures, and can often explain protein functionality in the context of structure. We herein report the systematic analysis of the local structures of proteins interacting with water molecules, and the characterization of their geometric features. We first examined the interaction of water molecules with a large local interaction environment by comparing the preference of water molecules in three regions, namely, the protein–protein interaction (PPI) interfaces, the crystal contact (CC) interfaces, and the non‐interfacial regions. High preference of water molecules to the PPI and CC interfaces was found. In addition, the bound water on the PPI interface was more favorably associated with the complex interaction structure, implying that such water‐mediated structures may participate in the shaping of the PPI interface. The pairwise water‐mediated interaction was then investigated, and the water‐mediated residue–residue interaction potential was derived. Subsequently, the types of polar atoms surrounding the water molecules were analyzed, and the preference of the hydrogen bond acceptor was observed. Furthermore, the geometries of the structures interacting with water were analyzed, and it was found that the major structure on the protein surface exhibited planar geometry rather than tetrahedral geometry. Several previously undiscovered characteristics of water–protein interactions were unfolded in this study, and are expected to lead to a better understanding of protein structure and function. Proteins 2016; 84:43–51. © 2015 Wiley Periodicals, Inc.     

Computational Analysis of Phosphopeptide Binding to the Polo-Box Domain of the Mitotic Kinase PLK1 Using Molecular Dynamics Simulation

The Polo-Like Kinase 1 (PLK1) acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD). The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors.     

Interfacial behavior of cytoplasmic and mitochondrial creatine kinase oligomeric states

Adsorption to the air/water interface of isoenzymes of creatine kinase was investigated using surface pressure-area isotherms and Brewster angle microscopy (BAM) observations. Octameric mitochondrial creatine kinase (mtCK) exhibits a significant affinity for the air/water interface. Whatever the mode of formation of the interfacial film, i.e., injection of the protein in the subphase or spreading onto the buffer surface, the final arrangement and conformation adopted by mtCK molecules lead to a similar result. In contrast, the dimeric isoenzymes mtCK and cytosolic MMCK do not induce any surface pressure variation. However, when the subphase contains 0.3M NaCl, both isoenzymes adsorb to the interface. When treated with 0.8 or 3M GdnHCl, muscle creatine kinase (MMCK) becomes surface active and occupies a greater surface than mtCK. This result contrasts with previous observations, often derived from monomeric proteins, that their surface activity is increased upon unfolding. It underlines the possible influence exerted by the protein oligomeric state on its interfacial activity. At a subphase pH of 8.8, which corresponds to the pI of octameric mtCK, the profiles of the isotherms obtained with dimeric and octameric states and the resistance to compression of the protein monolayers are significantly affected when compared to those recorded at pH 7.4. These data suggest that the octamer is more hydrophobic than the dimer and may contribute to explaining why octamers bind to the inner mitochondrial membrane while dimers do not.     

Imaging the ordered arrays of water-soluble protein ferritin with the atomic force microscope

Individual water-soluble molecules of the protein ferritin have been imaged on a silicon surface in pure water at room temperature with the atomic force microscope (AFM). The ferritin molecules formed an ordered monolayer by binding to a charged polypeptide monolayer of poly-1-benzyl-L-histidine (PBLH) spread at the air-water interface. The film, fully wetted with water, was horizontally transferred onto an alkylated silicon wafer for AFM imagings. The hexagonal arrangement of ferritin molecules was imaged with high reproducibility on the whole surface of the film, since the forces between cantilever and the sample could be kept sufficiently smaller than 10^-10 N, mainly due to a “self-screening effect” of the surface charges of the ferritin-PBLH layer. This is the first observation of two-dimensional ordered arrays of water-soluble protein molecules directly confirmed by AFM with molecular resolution.     

Driving forces of protein association: the dimer-octamer equilibrium in arylsulfatase A

The enzyme arylsulfatase A (ASA) occurs in solution as dimer (alpha(2)) above pH 6 and associates to octamers (alpha(2))(4) below pH 6. The crystal structure of ASA suggests that the (alpha(2))-(alpha(2))(4) equilibrium is regulated by protonation/deprotonation of Glu-424 located at the interface between (alpha(2)) dimers in the octamer. The reason for this assumption is that Glu-424 can be in two different conformers where it forms an intra or intermolecular hydrogen bond, respectively. In the present study we investigate this protein association process theoretically. The electrostatic energies are evaluated by solving the Poisson-Boltzmann equation for the inhomogeneous dielectric of the protein-water system for the dimer and octamer configurations. If a conventional surface energy term is used for the nonelectrostatic interactions, the absolute value of free energy of association fails to agree with experiment. A more detailed treatment that explicitly accounts for hydrophilic and hydrophobic character of the amino acids in the dimer-dimer interface of the octamer can explain this discrepancy qualitatively. The pH dependence of the computed association energy clearly demonstrates that the octamer is more stable at low pH if Glu-424 becomes protonated and forms an intermolecular hydrogen bond. We found a slight preference of Glu-424 to be in a conformation where its acidic group is fully solvent-exposed in the dimer state to form hydrogen bonds with water molecules. Application of the proton linkage model to calculate the association energy from the simulated data yielded results identical to the one obtained from the corresponding direct method.