A considerable amount (approximately 1.6 W Wg from 1 mg of dried nematode) of non-sulfated chondroitin, two orders of magnitude less yet an appreciable amount of heparan sulfate, and no hyaluronate were found in Caenorhabditis elegans nematodes. The chondroitin chains were heterogeneous in size, being shorter than that of whale cartilage chondroitin sulfate. The disaccharide composition analysis of heparan sulfate revealed diverse sulfation including glucosamine 2-N-sulfation, glucosamine 6-O-sulfation and uronate 2-O-sulfation. These results imply that chondroitin and heparan sulfate are involved in fundamental biological processes.z 1999 Federation of European Biochemical Societies.

To study the role of hydrogen bonding via interfacial water molecules in protein-protein interactions, we examined the interaction between hen egg white lysozyme (HEL) and its HyHEL-10 variable domain fragment (Fv) antibody. We constructed three antibody mutants (L-Y50F, L-S91A, and L-S93A) and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the mutations significantly decreased the negative enthalpy change (8 -25 kJ mol ؊1 ), despite some offset by a favorable entropy change. X-ray crystallography demonstrated that the complexes had nearly identical structures, including the positions of the interfacial water molecules. Taken together, the isothermal titration calorimetric and x-ray crystallographic results indicate that hydrogen bonding via interfacial water enthalpically contributes to the Fv-HEL interaction despite the partial offset because of entropy loss, suggesting that hydrogen bonding stiffens the antigen-antibody complex.Specific recognition of ligands by proteins is fundamentally important in biological phenomena. Recent high resolution xray crystallographic determinations of the structures of various protein-protein complexes have shown that protein-protein interfaces are generally characterized by a high degree of both shape and charge complementarity (1-3). The specificity and affinity of protein-protein interactions are in principle created by the complementarity of the interface surfaces, which allows the formation of various noncovalent bonds (hydrogen bonds, salt bridges, and van der Waals interactions) (1-3). Solvent water molecules, however, have been observed at protein-protein interfaces, mediating imperfect surface complementarity via hydrogen bond formation (1, 4 -7).Solvent plays a significant role in biologically important protein association systems (8), e.g. the adhesion interface of the cell adhesion protein cadherin (9), the barnase-barstar complex (5), the cytochrome c-cytochrome c oxidase interaction (10, 11), antigen-antibody interactions (12-16), the bacterial periplasmic protein (OppA)-peptide interaction (17, 18), the DNA-repressor interaction (19,20), the interaction between T-cell receptor and self-peptide major histocompatibility complex antigen (21), and the natural killer cell receptor-major histocompatibility complex antigen interaction (22). Ladbury (5) proposed that water at the interface of a complex can participate in various types of interaction, yet can also lead to increased specificity and affinity. However, the energetic contribution of hydrogen-bonded interfacial water molecules to protein-protein interactions has been determined for only a few complexes (18,23).Elucidation of the roles of the hydrogen bonds involved in antigen-antibody complementary association requires both structural and thermodynamic information (24). X-ray crystal analysis can clarify the structural aspects of the complementarity of the interactions (25-28), and titration calorimetry can provide useful information for the quant...

Abstract

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine-and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/ M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k cat value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-Å resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.To freely design enzymes with desired properties is the ultimate dream for protein engineers. Because the "protein folding problem," namely how an amino acid sequence determines its tertiary folded structure, has not completely been solved at atomic resolution, the rational design approach is still limited, even for improvement of enzymes from natural sources. Alternatively, protein design can be thought of as a process of "picking up" from all the possible amino acid sequences (i.e. sequence space) until a protein with the desired properties is found, because the structure and function of a protein is determined by its amino acid sequence (1). When we set the goal of protein design "to create a protein of a desired property consisting of a given number of amino acid residues n," the solution can be obtained by the complete search of all the possible amino acid sequences with "n" amino acid residues, the total number reaching 20 n . Therefore, the protein design problem can be reduced to a searching problem in sequence space of polypeptides with n amino acid residues. The solution to this searching problem should include a reliable process that can be performed within a realistic time span, otherwise it is of no use in practical protein design. In this regard, the size of sequence space to be searched is a critical factor (2). The size of the sequence space of a polypeptide with amino acids even as small as 100 is ...

A structural and thermodynamic study of the entropic contribution of salt bridge formation to the interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wildtype Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the antigen-antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein antigen-antibody interaction through reduction of entropy loss due to dehydration and structural changes.The ability of proteins to bind to one another in a highly specific manner is an important feature of biological phenomena, and the mechanism of antibody specificity and affinity toward target proteins has been studied as a model of proteinprotein interactions (1). X-ray crystal structures of antibodies complexed with proteinaceous antigens (2-6) suggest that the strict specificity of an antibody originates from a high degree of complementarity between the antigen-combining sites and the antigen. The complementarity is composed of two factors: 1) apolar surfaces interacting by van der Waals contact, where the surfaces of the antibody and antigen often fit together to exclude most hydrated water molecules from the interface and favorable close van der Waals contacts are formed; and 2) polar surfaces interacting by hydrogen bond and salt bridge formation, where the precise positioning of atoms allows chargecharge interactions (i.e. salt bridge formation and hydrogen bond formation).To elucidate the roles of the noncovalent forces involved in antigen-antibody complementary association, both structural and thermodynamic information are crucial (7). X-ray crystal analysis can clarify the structural aspects for the complementarity of the interactions (2-5), and titration calorimetry can provide useful information for the quantitative assessment of the contribution of residues to the interaction (8 -10). Thus, the combination of these...