Interplay of Local Structure, Dynamics, and Self-Association in HSPB1
Clouser, Amanda Faith
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Small heat shock proteins (sHSPs) comprise a class of ATP-independent chaperones that prevent aggregation of destabilized proteins in the cell. The structural mechanisms by which sHSPs complete their function remain enigmatic due to inherent properties that make them refractory to traditional approaches in structural biology. While sHSP monomers are small (on average 20 kDa), for certain sHSPs these monomers assemble into large homo-oligomers (~500 kDa) of variable size and shape. Structural heterogeneity is observed not only on a global scale but also at the local level. Observed heterogeneity is thought to stem from the predominantly disordered N-terminal region (NTR) of sHSPs. There is evidence for numerous sites on sHSPs for interacting with client proteins, although these interactions are often transient. The combination of transient interactions with clients, competing sHSP-client and sHSP-sHSP interactions, and presentation of different binding sites in variable states of an oligomer make it particularly challenging to understand structure-function relationships in sHSPs. The work presented in this thesis correlates local and global structural properties of the polydisperse human sHSP HSPB1. Hydrogen-deuterium exchange mass spectrometry (HDXMS) was used to identify solvent protected regions in the NTR of HSPB1 oligomers, providing the first full look at the NTR of an oligomeric human sHSP. Additionally, specific regions of local structural heterogeneity were identified in the NTR. Utilizing known mutations that perturb inter-protomer interactions in oligomers, a dimeric, full-length form of HSPB1 was obtained to represent a dispersed subunit of the system. The dimeric form of HSPB1 shows altered local structure in the NTR by HDXMS relative to WT oligomers, indicating distinct structural changes among different states of HSPB1. The dimeric construct is also amenable to traditional NMR approaches, allowing for development of a comprehensive model of full-length HSPB1 dimers, which still show structural heterogeneity. Disease-associated mutations in the NTR of HSPB1 were also characterized using HDXMS and a suite of other biophysical techniques. Specific regions in the NTR have altered protection among these disease mutants. These disease mutations were introduced to the dimeric construct to probe each mutation’s effect on oligomerization propensity. Interestingly, two of the disease mutations promote oligomerization while the other two disease mutations retain some dimer-like characteristics in the dimer-mutant context. HDXMS analysis of the disease mutants in the dimeric construct reveals distinct local structural effects for each mutant. It is most remarkable that mutations in the same region of HSPB1 have different local mechanistic effects, and that the subsequent global effects on oligomeric size or chaperone activity can appear similar for certain mutants. The methods explored in this thesis for characterizing heterogeneous structure in HSPB1 can be implemented to other sHSPs and similarly complex protein systems.
- Biological chemistry