CONFORMATIONAL STUDIES OF PROTEINS IN CONTACT ACTIVATION

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The long term goal of this project is to understand the mechanism that are responsible for the expression of biological activities of each of the four proteins involved in the contact phase of blood coagulation. The conformational nad structural aspects of their interactions will be investigated using various biophysical methods, such as circular dichroism, solvent perturbation, uv difference spectroscopy, light scattering, sedimentation, chemical modification, molecular model building and prediction of protein secondary structure. Specifically, the objectives will be: (1) Peptide sequences in factor XII which are known to be involved in surface binding will be synthesized. These peptides, which correspond to factor Xiii-28 and factor XII134-153 of the intact factor XII will be modified by amino acid substitution and specific chemical modifications. Systematic kinetic and conformational analysis will be undertaken to determine how various forms of modifications of the peptides will affect the inhibition of factor XII activation by negatively charged surfaces. (2) Peptides containing the GHKHER and HGLGHGH sequences in HMWK which are known to be involved in surface binding will be synthesized. These in HMWK which are known to be involved in surface binding will be synthesized. These unique sequences, which correspond to HMWK426-431, HMWK441-447 and HMWK451-457 of the intact HMWK will be modified by amino acid substitution and specific chemical modifications. Systematic kinetic and conformational analysis will be conducted to investigate how various alterations in charges and amino acid substitutions will affect the ability of the peptides to inhibit HMWK binding to PK and factor XI will be synthesized. These peptides, which correspond to HMWK556-595 and HMWK556-613 in the intact HMWK will be modified by amino acid substitution and chemical modifications. Systematic kinetic and conformational analysis will be undertaken to investigate how various ways of modifications of the peptides will affect the ability to inhibit the binding of HMWK to the two zymogen. (4) Finally, to use chemical crosslinking reagent, in conjunction with the synthetic peptides, HMWK556-595 and HMWK556-613, in order to establish and define the HMWK binding site in PK and factor XI.