Large Protein Folding and Dynamics Studied by Advanced Hydrogen Exchange Methods

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Degree type
Doctor of Philosophy (PhD)
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Biochemistry & Molecular Biophysics
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Denatured State Ensemble DSE
HDX MS
Hydrogen Exchange
Maltose Binding Protein
Protein Folding
Analytical Chemistry
Biochemistry
Biophysics
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2014-08-22T20:13:00-07:00
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Abstract

Protein folding studies over the past 50 years have been largely focused on small proteins (< 200 residues) leading to a dearth of information about large protein folding. Regardless of protein size, research has generally lacked the structural tools with necessary temporal resolution to provide mechanistic insight into the process. This goal requires incisive information on transient kinetic intermediate conformations that describe the folding pathway. In this work special challenges that hinder large protein folding studies are addressed, and advancements to both HX NMR and HX MS experiments are described that provide unparalleled temporal resolution of structure formation than has been previously possible. These various advanced hydrogen exchange methods are used to study folding behaviors of the large, 370-residue, two-domain maltose binding protein from E. coli and provide a description of its folding pathway in structural detail. This work sheds light on two basic unresolved problems regarding the mechanisms of protein folding, the first being the enigmatic nature of the initial folding collapse event seen in many proteins, and the second concerning the nature of the folding pathway. We find that from an initially heterogeneous hydrophobic collapse, an obligatory intermediate emerges with a 7-second time constant followed by an apparent sequential pathway to the native state. These results add the largest protein studied at structural resolution to-date to the list of proteins known to fold through obligatory, native-like intermediates in distinct pathways and this work highlights strategies that may be employed to interrogate other large systems in future work.

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S. Walter Englander
Feng Gai
Date of degree
2013-01-01
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