Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Gary Shaw

Abstract

S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by ANS fluorescence and phenyl Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target-binding site. In contrast, S100A11 chimeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and consequently, unobservable annexin A2 binding. This work represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10.