We report the three-dimensional structures, at 1.8-A resolution, ofthe Fv fragment ofthe anti-hen egg white lysozyme antibody D1.3 in its free and antigen-bound forms.These structures reveal a role for solvent molecules in stabilzing the complex and provide a molecular basis for understanding the thermodynamic forces which drive the association reaction. Four water molecules are buried and others form a hydrogen-bonded network around the interface, bridging antigen and antibody. Comparison of the structures of free and bound Fv fragment of D1.3 reveals that several of the ordered water molecules in the free antibody combining site are retained and that additional water molecules link antigen and antibody upon complex formation. This salvation of the complex should weaken the hydrophobic effect, and the resulting large number of solvent-mediated hydrogen bonds, in conijunction with direct protein-protein interactions, should generate a significant enthalpic component. Furthermore, a stabilization of the relative mobilities of the antibody heavy-and light-chain variable domains and of that of the third complementaritydetermining loop of the heavy chain seen in the complex should generate a negative entropic contribution opposing the enthalpic and the hydrophobic (solvent entropy) effects. This structural analysis is consistent with measurements of enthalpy and entropy changes by titration calorimetry, which show that enthalpy drives the antigen-antibody reaction. Thus, the main forces stabilizing the complex arise from antigen-antibody hydrogen bonding, van der Waals interactions, enthalpy of hydration, and conformational stabilization rather than solvent entropy (hydrophobic) effects.X-ray crystallographic studies of several complexes of antigens with specific antibodies have revealed a high degree of complementarity between their interacting surfaces (reviewed in refs. 1 and 2). Water molecules have been identified at the interfaces of the Fab fragment of antibody D1.3 (Fab D1.3)-hen egg-white lysozyme (HEL) (3) and NC41-neuraminidase (4) complexes on the basis of structure determinations at 2.5-A resolution. Unfortunately, at such resolution, which is about the best which has been so far attained with conventional Fab fragments, the certainty with which ordered water molecules can be located is seriously limited (5). We have now determined the three-dimensional structure, at 1.8-A resolution, of the Fv fragment of monoclonal antibody (mAb) D1.3 (6, 7), Fv D1.3, consisting of only the variable domains ofthe heavy (VH) and light (VL) polypeptide chains and that of its complex with HEL, permitting a more detailed description of an antibody combining site in its free and antigen-bound states. These studies reveal both buried and exposed water molecules linking antigen and antibody and contributing to chemical complementarity between their interacting surfaces.An understanding of how antibodies react with antigens must involve the thermodynamics of the binding interaction. We have therefore experimentally de...

Titration calorimetry was used to measure equilibrium constants and standard molar enthalpies for the reactions of phenethylamine, ephedrines, and related substances with a-and /3-cyclodextrin. Changes in the chemical shifts A6 of both the ligand and cyclodextrin protons were measured with NMR. The thermodynamic results have been examined in terms of structural features of the ligand that affect these interactions such as the separation of the charge at an amino group and the aromatic ring, steric effects, the presence of additional functional groups (amino, hydroxy, methoxy, and methyl) attached to the aromatic ring, the presence and location of hydroxy group(s) on the ligand, changes in the chirality of the ligand, and the flexibility of the organic molecules attached to the aromatic ring. It was found that the values of thermodynamic quantities for these reactions in phosphate and acetate buffers were different. This difference is attributable to the presence of a hydrophobic alkyl group in the neutral acetic acid molecule and its interaction with the cyclodextrins. Also, there are significant differences in the thermodynamic quantities for the reactions of the chiral isomers of ephedrine and pseudoephedrine in their reactions with /3-cyclodextrin. A plot of the standard molar enthalpy vs the standard molar entropy for the reactions of these chiral isomers with a-and /3-cyclodextrin is linear; the relative order of the ephedrines and pseudoephedrines in the enthalpy-entropy plot is the same for the reactions of these substances with both a-and /3-cyclodextrin. NMR studies demonstrated that the magnitude of the upfield shifts of the cyclodextrin's H3 and H5 protons, A<5(H3) and A<5(H5), and their relative ratio, A<5(H5)/A<5(H3), can be used, respectively, as a measure of the complex stability and the depth of inclusion of the ligand into the cavity. The equilibrium constants determined by titration calorimetry correlate well with the changes in chemical shifts Ad determined by NMR.

No abstract

Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25 degrees C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25 degrees C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B).(ABSTRACT TRUNCATED AT 250 WORDS)

This work is concerned with the accurate quantification of brain water content under routine clinical conditions. Gelatin solutions of varying water content are first employed as a model of an edematous brain and longitudinal relaxation measurements are performed at proton Larmor frequencies of 5, 41, 63, and 100 MHz. These are followed with in vivo measurements in an experimental animal model of brain edema at 41 MHz. The results underscore the dominant role of total water content W in the relaxation process and verify the expected linearity between 1/T1 and 1/W. A scheme is presented and experimentally verified at 41 MHz for deducing the exact relationship of 1/T1 vs 1/W at any frequency. Knowledge of this relationship along with precise measurements of 1/T1 at a given field strength permits quantitative in vivo measures of brain water content to be obtained with a precision of about 0.01. It is concluded that routine, accurate, and noninvasive brain water measurements are possible by magnetic resonance imaging in a clinical environment.