Masters Theses

Date of Award

12-2003

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Chris Dealwis

Abstract

Resulting from a high degree of sequence variability, a few human light-chains have a propensity to form amyloid fibrils. The monoclonal V J6 proteins are involved in fibril formation resulting in amyloidosis (AL). A previous study of a· patient (Jto) with multiple myeloma in whom the V J6 protein was deposited in the form of renal tubular casts was shown to form fibrils very slowly. The slow fibril formation resulted from the improved thermodynamic stability gained from an extra salt-bridge between Asp 29 and Arg 66B. To further investigate the effect of this interaction, two mutants Asp 29 to an Ala (JtoD29A) and Arg 66B to a Ser (JtoR68S) that disrupts the salt-bridge were made. Interestingly, the JtoD29A and JtoR68S have very different kinetics of fibril formation. The JtoD29A forms fibrils very slowly, while the JtoR68S forms fibrils at a rate similar to the pathogenic V).6 Wil. In this study we have crystallized JtoD29A and JtoR68S in the same space group P4122 with the same cell dimensions and solved their X-ray structures to 1.6 A and 1.9 A resolution, respectively. Structural comparisons reveal that although there are no significant main-chain conformational changes, there are several side-chain conformational changes due to the mutations. These differences contribute to JtoR68S having a larger exposed hydrophobic solvent surface as compared to JtoD29A. The Arg to Ser mutation in JtoR68S is also responsible for its increased negative electrostatic potential as compared to JtoD29A. We have also attempted to trap a pH induced structural intermediate of JtoR68S. While these findings are preliminary and need to be further investigated, these methods provide new means and insights for studying the amyloid phenomenon.

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