Development of dynamical structural analysis for the active site of multi functional proteins

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In the present study, we develop the NMR method in order to analyze the dynamical structure of the active site of multi-functional proteins. The questions to resolve are1) How do we selectively obtain the information about the structure of the active site of the multi-functional protein?2) How do we analyze the dynamical structure of the protein in solution?The answere are the use of protein which are amino-acid specifically labeled withe stable isotope and the analysis of the relaxation time of the amide group.The following results are obtained from the present study.Study by using of anti-dansvl Fv fragment1) The assignments of the signals originating from the amide group of the main chain of anti-dansyl Fv fragment are established by using the double labeling method along with the sequence-specific resonance assingment.2) On the basis of the results obtained from the experiment of the binding of the spin labeled hapten, we conclude that the antigen-binding site of the Fv fragment is composed of H1, H3 of the hypervariable loops and N-terminal in VH domain.3) On the basis of the NOE data, we conclude that the residues which are responsible for the antigen binding are Y96H,Y104H,F27H and V2H.4) The effect of the antigen binding is transmitted from VH domain to VL domain through the interface of the Fv fragment.5) In the absence of the antigen, the hyper variable loop which is responsible for the antigen binding take multi-conformations. The exchange rate among the conformations is affected by the antigen binding.6) From the comparison of the structure of the antigen binding site between in the absence and presence of the antigen, the mechanism of the antigen binding for the Fv fragment is 'induced fit'.Study by using of anti-dansyl Fab fragmentThe method established by using the Fv fragment with the molecular weight of 25K is able to apply to the Fab fragment with the molecular weight of 50K.