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Author ORCID Identifier

N/A

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2018

Month Degree Awarded

May

First Advisor

Elizabeth Vierling

Second Advisor

Peter Chien

Third Advisor

Stephen Eyles

Fourth Advisor

Igor Kaltashov

Subject Categories

Biochemistry | Cell Biology | Molecular Biology

Abstract

Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to protein misfolding diseases. They comprise a conserved core α-crystallin domain (ACD) flanked by an evolutionarily variable N-terminal domain (NTD) and semi-conserved C-terminal extension/domain (CTD). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that coordinate with ATP-dependent chaperones for refolding. To derive common features of sHSP-substrate recognition, I compared the chaperone activity and specific sHSP-substrate interaction sites for three different sHSPs from Arabidopsis (At17.6B), pea (Ps18.1) and wheat (Ta16.9), for which the atomic solution-state structures were modeled. I used the homobifunctional, amine- reactive, 7 Å cross-linker bis(sulfosuccinimidyl)glutarate (BS2G) to report on interaction sites between the sHSPs, a model substrate, malate dehydrogenase (MDH), and interactions between At17.6B and an endogenous target, fructose-1,6-bisphosphatealdolase (FBA). Interaction sites were identified by examining the position of residues that were modified by BS2G and coordinated cross-links with residues in other peptides, which were detected using ESI-LC-MS/MS. MDH exhibited major molecular rearrangement upon heat denaturation and association with sHSPs evidenced by detection of new MDH-MDH cross-links incompatible with the native structure. The NTD of all three sHSPs was a major site of cross-linking to substrate and was found linked to multiple sites on MDH, consistent with heterogeneous molecular interactions. While the N-terminus of Ta16.9 and At17.6B participated in many sHSP-MDH interactions, the more efficient Ps18.1 cross- linked MDH at multiple sites using all three sHSP domains. Not all sHSP or MDH Lys residues formed cross-links, despite displaying reactivity with the cross-linker, demonstrating preferred sites of interaction. At17.6B-FBA interactions closely resembled sHSP-model substrate data acquired with MDH, validating the cross-linking method and interaction models. Both BS2G and the UV-inducible, site specific cross-linker benzoylphenylalanine (Bpa), were used to detect direct sHSP-Hsp70 interactions during the substrate refolding process, suggesting that inter-chaperone interaction may play a role in the mechanismrequired for substrate handoff from the sHSP-substrate complex to Hsp70 for refolding.

DOI

https://doi.org/10.7275/11948670

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