Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins.The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.     

Apolar and polar solvation thermodynamics related to the protein unfolding process.

Thermodynamics related to hydrated water upon protein unfolding is studied over a broad temperature range (5-125 degrees C). The hydration effect arising from the apolar interior is modeled as an increased number of hydrogen bonds between water molecules compared with bulk water. The corresponding contribution from the polar interior is modeled as a two-step process. First, the polar interior breaks hydrogen bonds in bulk water upon unfolding. Second, due to strong bonds between the polar surface and the nearest water molecules, we assume quantization using a simplified two-state picture. The heat capacity change upon hydration is compared with model compound data evaluated previously for 20 different proteins. We obtain good correspondence with the data for both the apolar and the polar interior. We note that the effective coupling constants for both models have small variations among the proteins we have investigated.     

Electrostatic contributions to heat capacity changes of DNA-ligand binding.

Significant heat capacity changes (DeltaCp) often accompany protein unfolding, protein binding, and specific DNA-ligand binding reactions. Such changes are widely used to analyze contributions arising from hydrophobic and polar hydration. Current models relate the magnitude of DeltaCp to the solvent accessible surface area (ASA) of the molecule. However, for many binding systems-particularly those involving non-peptide ligands-these models predict a DeltaCp that is significantly different from the experimentally measured value. Electrostatic interactions provide a potential source of heat capacity changes and do not scale with ASA. Using finite-difference Poisson-Boltzmann methods (FDPB), we have determined the contribution of electrostatics to the DeltaCp associated with binding for DNA binding reactions involving the ligands DAPI, netropsin, lexitropsin, and the lambda repressor binding domain.     

Solid model compounds and the thermodynamics of protein unfolding.

Analysis of thermodynamic data on the dissolution of solid cyclic dipeptides into water in terms of group additivity provides a rationale for the enthalpy and entropy convergence temperatures observed for small globular protein denaturation and the dissolution of model compounds into water. Convergence temperatures are temperatures at which the extrapolated enthalpy or entropy changes for a series of related compounds take on a common value. At these temperatures (TH* and TS*) the apolar contributions to the corresponding thermodynamic values (delta H degrees and delta S degrees) are shown to be zero. Other contributions such as hydrogen bonding and configurational effects can then be evaluated and their quantitative effects on the stability of globular proteins assessed. It is shown that the denaturational heat capacity is composed of a large positive contribution from the exposure of apolar groups and a significant negative contribution from the exposure of polar groups in agreement with previous results. The large apolar contribution suggests that a liquid hydrocarbon model of the hydrophobic effect does not accurately represent the apolar contribution to delta H degrees of denaturation. Rather, significant enthalpic stabilizing contributions are found to arise from peptide groups (hydrogen bonding). Combining the average structural features of globular proteins (i.e. number of residues, fraction of buried apolar groups and fraction of hydrogen bonds) with their specific group contributions permits a first-order prediction of the thermodynamic properties of proteins. The predicted values compare well with literature values for cytochrome c, myoglobin, ribonuclease A and lysozyme. The major thermodynamic features are described by the number of peptide and apolar groups in a given protein.     

Structural energetics of MgADP binding to the isolated beta subunit of F1-ATPase from thermophilic Bacillus PS3

The energetics of binding of MgADP to the isolated beta subunit of F(1)-ATPase from thermophilic Bacillus (Tbeta) was characterized by high-precision isothermal titration calorimetry. The reaction was enthalpically driven, with a DeltaCp of -36cal(molK)(-1). To gain insight into the molecular basis of this small DeltaCp, we analyzed the changes in accessible surface areas (DeltaASA) between the structures of empty and MgADP-filled beta subunits, extracted from the crystal structure of bovine heart F(1). Consistent with the experimental DeltaCp, the DeltaASA was small (-775A(2)). We used a reported surface area model developed for protein reactions to calculate DeltaCp and DeltaH from DeltaASA, obtaining good agreement with the experimental values. Conversely, using the same model, a DeltaASA of -770A(2) was estimated from experimental DeltaCp and DeltaH for the Tbeta-MgADP complex. Our structural-energetic study indicates that on MgADP binding the isolated Tbeta subunit exhibits intrinsic structural changes similar to those observed in F(1).     

Estimation of the hydrophobic effect in an antigen-antibody protein-protein interface

Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V(L)W92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a peptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fold lower than that of the original antibody. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92 mutant series and changes in the enthalpy of formation. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R(2) = 0.937) as well as increases in the solvent portion of the entropy of binding (R(2) = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistent with values for the hydrophobic effect derived from classical hydrocarbon solubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.     

The mechanism of the type III antifreeze protein action: a computational study

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.     

Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.     

Modeling protein recognition of carbohydrates

We have a limited understanding of the details of molecular recognition of carbohydrates by proteins, which is critical to a multitude of biological processes. Furthermore, carbohydrate-modifying proteins such as glycosyl hydrolases and phosphorylases are of growing importance as potential drug targets. Interactions between proteins and carbohydrates have complex thermodynamics, and in general the specific positioning of only a few hydroxyl groups determines their binding affinities. A thorough understanding of both carbohydrate and protein structures is thus essential to predict these interactions. An atomic-level view of carbohydrate recognition through structures of carbohydrate-active enzymes complexed with transition-state inhibitors reveals some of the distinctive molecular features unique to protein-carbohydrate complexes. However, the inherent flexibility of carbohydrates and their often water-mediated hydrogen bonding to proteins makes simulation of their complexes difficult. Nonetheless, recent developments such as the parameterization of specific force fields and docking scoring functions have greatly improved our ability to predict protein-carbohydrate interactions. We review protein-carbohydrate complexes having defined molecular requirements for specific carbohydrate recognition by proteins, providing an overview of the different computational techniques available to model them.     

Adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates.

Isothermal titration calorimetry (ITC) was used to test the hypothesis that the relatively small enthalpy change (DeltaHobs) and large negative heat capacity change (DeltaCp,obs) observed for the binding of the Escherichia coli SSB protein to single-stranded (ss) oligodeoxyadenylates result from the temperature-dependent adenine base unstacking equilibrium that is thermodynamically coupled to binding. We have determined DeltaH1,obs for the binding of 1 mole of each of dT(pT)34, dC(pC)34, and dA(pA)34 to the SSB tetramer (20 mM NaCl at pH 8.1). For dT(pT)34 and dC(pC)34, we found large, negative values for DeltaH1,obs of -75 +/- 1 and -85 +/- 2 kcal/mol at 25 degrees C, with DeltaCp,obs values of -540 +/- 20 and -570 +/- 30 cal mol-1 K-1 (7-50 degrees C), respectively. However, for SSB-dA(pA)34 binding, DeltaH1,obs is considerably less negative (-14 +/- 1 kcal/mol at 25 degrees C), even becoming positive at temperatures below 13 degrees C, and DeltaCp,obs is nearly twice as large in magnitude (-1180 +/- 40 cal mol-1 K-1). These very different thermodynamic properties for SSB-dA(pA)34 binding appear to result from the fact that the bases in dA(pA)34 are more stacked at any temperature than are the bases in dC(pC)34 or dT(pT)34 and that the bases become unstacked within the SSB-ssDNA complexes. Therefore, the DeltaCp,obs for SSB-ssDNA binding has multiple contributions, a major one being the coupling to binding of a temperature-dependent conformational change in the ssDNA, although SSB binding to unstacked ssDNA still has an "intrinsic" negative DeltaCp,0. In general, such temperature-dependent changes in the conformational "end states" of interacting macromolecules can contribute significantly to both DeltaCp,obs and DeltaHobs.     

Hydrophobic tendency of polar group hydration as a major force in type I antifreeze protein recognition

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of distortions of the water-water hydrogen bonding angle in the first hydration shell (Gallagher and Sharp, JACS 2003;125:9853). The distribution of this angle in pure water is bimodal, with a low-angle population and high-angle population. Polar solutes increase the high-angle population while apolar solutes increase the low-angle population. The ratio of the two populations quantifies the hydrophobicity of the solute and provides a sensitive measure of water structural distortions. This method of analysis is applied to study hydration of type I thermal hysteresis protein (THP) from winter flounder and three quadruple mutants of four threonine residues at positions 2, 13, 24, and 35. Wild-type and two mutants (VVVV and AAAA) have antifreeze (thermal hysteresis) activity, while the other mutant (SSSS) has no activity. The analysis reveals significant differences in the hydration structure of the ice-binding site. For the SSSS mutant, polar groups have a typical polar-like hydration, that is, more high-angle H-bonds than bulk water. For the wild-type and active mutants, polar groups have unusual, very apolar-like hydration, that is, more low-angle H-bonds than bulk water. This pattern of hydration was seen previously in the structurally distinct type III THPs (Yang & Sharp Biophys Chem 2004;109:137), suggesting for the first time a general mechanism for different THP classes. The specific shape, residue size, and clustering of both polar and apoler groups are essential for an active ice binding surface.     

Detection of specific solvent rearrangement regions of an enzyme: NMR and ITC studies with aminoglycoside phosphotransferase(3')-IIIa

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.     

Temperature dependence and thermodynamics of Klenow polymerase binding to primed-template DNA

DNA binding of Klenow polymerase has been characterized with respect to temperature to delineate the thermodynamic driving forces involved in the interaction of this polymerase with primed-template DNA. The temperature dependence of the binding affinity exhibits distinct curvature, with tightest binding at 25-30 degrees C. Nonlinear temperature dependence indicates Klenow binds different primed-template constructs with large heat capacity (DeltaCp) values (-870 to -1220 cal/mole K) and thus exhibits large temperature dependent changes in enthalpy and entropy. Binding is entropy driven at lower temperatures and enthalpy driven at physiological temperatures. Large negative DeltaCp values have been proposed to be a 'signature' of site-specific DNA binding, but type I DNA polymerases do not exhibit significant DNA sequence specificity. We suggest that the binding of Klenow to a specific DNA structure, the primed-template junction, results in a correlated thermodynamic profile that mirrors what is commonly seen for DNA sequence-specific binding proteins. Klenow joins a small number of other DNA-sequence independent DNA binding proteins which exhibit unexpectedly large negative DeltaCp values. Spectroscopic measurements show small conformational rearrangements of both the DNA and Klenow upon binding, and small angle x-ray scattering shows a global induced fit conformational compaction of the protein upon binding. Calculations from both crystal structure and solution structural data indicate that Klenow DNA binding is an exception to the often observed correlation between DeltaCp and changes in accessible surface area. In the case of Klenow, surface area burial can account for only about half of the DeltaCp of binding.     

A model binding site for testing scoring functions in molecular docking

Prediction of interaction energies between ligands and their receptors remains a major challenge for structure-based inhibitor discovery. Much effort has been devoted to developing scoring schemes that can successfully rank the affinities of a diverse set of possible ligands to a binding site for which the structure is known. To test these scoring functions, well-characterized experimental systems can be very useful. Here, mutation-created binding sites in T4 lysozyme were used to investigate how the quality of atomic charges and solvation energies affects molecular docking. Atomic charges and solvation energies were calculated for 172,118 molecules in the Available Chemicals Directory using a semi-empirical quantum mechanical approach by the program AMSOL. The database was first screened against the apolar cavity site created by the mutation Leu99Ala (L99A). Compared to the electronegativity-based charges that are widely used, the new charges and desolvation energies improved ranking of known apolar ligands, and better distinguished them from more polar isosteres that are not observed to bind. To investigate whether the new charges had predictive value, the non-polar residue Met102, which forms part of the binding site, was changed to the polar residue glutamine. The structure of the resulting Leu99Ala and Met102Gln double mutant of T4 lysozyme (L99A/M102Q) was determined and the docking calculation was repeated for the new site. Seven representative polar molecules that preferentially docked to the polar versus the apolar binding site were tested experimentally. All seven bind to the polar cavity (L99A/M102Q) but do not detectably bind to the apolar cavity (L99A). Five ligand-bound structures of L99A/M102Q were determined by X-ray crystallography. Docking predictions corresponded to the crystallographic results to within 0.4A RMSD. Improved treatment of partial atomic charges and desolvation energies in database docking appears feasible and leads to better distinction of true ligands. Simple model binding sites, such as L99A and its more polar variants, may find broad use in the development and testing of docking algorithms.     

Thermodynamics of DNA binding and distortion by the hyperthermophile chromatin protein Sac7d

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.     

Break in the heat capacity change at 303 K for complex binding of netropsin to AATT containing hairpin DNA constructs

Studies performed in our laboratory demonstrated the formation of two thermodynamically distinct complexes on binding of netropsin to a number of hairpin-forming DNA sequences containing AATT-binding regions. These two complexes were proposed to differ only by a bridging water molecule between the drug and the DNA in the lower affinity complex. A temperature-dependent isothermal titration calorimetry (ITC)-binding study was performed using one of these constructs (a 20-mer hairpin of sequence 5'-CGAATTCGTCTCCGAATTCG) and netropsin. This study demonstrated a break in the heat capacity change for the formation of the complex containing the bridging water molecule at approximately 303 K. In the plot of the binding enthalpy change versus temperature, the slope (DeltaCp) was -0.67 kcal mol-1 K-1 steeper after the break at 303 K. Because of the relatively low melting temperature of the 20-mer hairpin (341 K (68 degrees C)), the enthalpy change for complex formation might have included some energy of refolding of the partially denatured hairpin, giving the suggestion of a larger DeltaCp. Studies done on the binding of netropsin to similar constructs, a 24-mer and a 28-mer, with added GC basepairs in the hairpin stem to increase thermal stability, exhibit the same nonlinearity in DeltaCp over the temperature range of from 275 to 333 K. The slopes (DeltaCp) were -0.69 and -0.64 kcal mol-1 K-1 steeper after 303 K for the 24-mer and 28-mer, respectively. This observation strengthens the argument regarding the presence of a bridging water molecule in the lower affinity netropsin/DNA complex. The DeltaCp data seem to infer that because the break in the heat capacity change function for the lower affinity binding occurs at the isoequilibrium temperature for water, water may be included or trapped in the complex. The fact that this break does not occur in the heat capacity change function for formation of the higher affinity complex can similarly be taken as evidence that water is not included in the higher affinity complex.     

Analysis of thermal hysteresis protein hydration using the random network model

The hydration of polar and apolar groups can be explained quantitatively, via the random network model of water, in terms of differential distortions in first hydration shell water-water hydrogen bonding angle. This method of analyzing solute induced structural distortions of water is applied to study the ice-binding type III thermal hysteresis protein. The analysis reveals subtle but significant differences in solvent structuring of the ice-binding surface, compared to non-ice binding protein surface. The major differences in hydration in the ice-binding region are (i). polar groups have a very apolar-like hydration. (ii). there is more uniform hydration structure. Overall, this surface strongly enhances the tetrahedral, or ice-like, hydration within the primary hydration shell. It is concluded that these two specific features of the hydration structure are important for this surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.     

Nuclear magnetic resonance and ultraviolet difference spectral studies of the binding properties of turkey egg white lysozyme. Consequences of the replacement of Asp 101 by glycine

The nuclear magnetic resonance spectrum of the ¹⁹F nuclei in N-trifluoroacetylated chitotriose was studied in the presence of turkey lysozyme. In contrast to results previously obtained with hen lysozyme, the ¹⁹F nmr spectrum of the complex did not show any striking pH dependence. It was, in fact, very similar at all pH's to the spectrum of the trisaccharide complexed with hen lysozyme at low pH, where Asp 101 is protonated. The replacement of Asp 101 in turkey lysozyme by a glycine is thought to account for this difference and the results allow unequivocal assignment of a value of 4.2 to the pK_a of Asp 101 in hen lysozyme. The dissociation constant of the chitotriose-turkey lysozyme complex was measured at various pH's using uv difference methods and compared with that previously reported for the hen lysozyme-chitotriose complex. Again, the results could be attributed to the loss in binding energy due to the absence of Asp 101. In contrast to chitotriose, the binding of chitobiose and methyl-2-acetamido-2-deoxy-β-d-glucopyranoside as studied by both uv difference and nmr methods is the same within experimental error for turkey and hen lysozyme. The results obtained for binding of chitobiose suggest that Asp 101 does not contribute as much to the binding energy of the disaccharide as was previously thought. Finally, the specific activities of both of these lysozymes against Micrococcus lysodeikticus were found to be identical.     

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).     

Sequential hydration of dry proteins: a direct difference IR investigation of sequence homologs lysozyme and alpha- lactalbumin

Direct difference ir spectra are presented as a function of hydration for lysozyme and α-lactalbumin, and detailed sequential hydration molecular events identified. Despite the strong sequence homology between the two proteins, and their expected conformational similarity, the hydration behaviour of the polar groups is different for the two proteins. Using a Hill-type analysis, we conclude that the acid groups ionize and hydrate rapidly and noncooperatively in both proteins, consistent with the known (lysozyme) and postulated (α-lactalbumin) surface chemistry. The polar group hydration shows a clear cooperativity, which is quantitatively different in the two proteins. Complementary work suggests this cooperativity relates to a hydration-induced “loosening up” of the lysozyme conformation at about 55 mol water/mol protein. α-Lactalbumin appears to “open up” more easily for hydration than does lysozyme, consistent with its lower stability against thermal and acid denaturation.