DETERMINATION OF FOLDING PATHWAY SELECTION AND ORIGINS OF COOPERATIVITY OF NATURALLY OCCURRING AND DESIGNED CONSENSUS LEUCINE-RICH REPEAT PROTEINS

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dc.contributor.advisorGarcia-Moreno, Bertrand E.en_US
dc.contributor.authorDao, Thuy Phuongen_US
dc.contributor.committeeMemberHilser, Vincent J.en_US
dc.contributor.committeeMemberLecomte, Juliette T. J.en_US
dc.contributor.committeeMemberTolman, Joel R.en_US
dc.date.accessioned2015-09-16T03:38:54Z
dc.date.available2015-09-16T03:38:54Z
dc.date.created2014-08en_US
dc.date.issued2014-07-16en_US
dc.date.submittedAugust 2014en_US
dc.description.abstractThermodynamic and kinetic studies of linear repeat proteins have provided unique insights into cooperativity, detailed maps of local stabilities, and sequence determinants of folding pathways. Most of these studies have focused on α helical repeat proteins. Additional work on repeat proteins that feature other types of secondary structures, such as β-strands, is essential for understanding different kinds of interactions found in much more complicated globular proteins. Here, we investigated the folding properties of a naturally occurring β strand containing leucine-rich repeat (LRR) protein PP32 as well as designed consensus bacterial LRR constructs. PP32 contains five tandem LRRs flanked by α-helical and β-strand capping motifs on the N- and C-termini, respectively. Terminal caps are often observed in LRR proteins, but not in helical repeat proteins. Without the C-cap, PP32 is unfolded. Without the N-cap, PP32 is less stable, but retains its secondary structure. However, solution studies by NMR and mutational analysis show that removing the N-cap causes the first two repeats to exist in a molten globule-like state, where secondary structures are formed but rigid tertiary packing is disrupted. Therefore, both caps are essential for structure formation and stability of PP32, though to different extents. Although PP32 undergoes an equilibrium two-state unfolding transition, its kinetic folding mechanism is more complicated, with the formation of an on pathway intermediate as the rate-limiting step. Φ-value analysis reveals a highly polarized transition state involving repeat 5 and part of repeat 4. Hydrogen exchange monitored by NMR spectroscopy shows that PP32 is most stable towards the C-terminus. Therefore, the folding pathway for PP32 is dictated by local stability, as observed for α-helical repeat proteins. Whereas the studies of naturally occurring repeat proteins permit the variations in repeat sequence to be investigated, those of designed consensus repeats proteins, in which the repeats are identical (or nearly identical), allow us to resolve the intrinsic energy of individual repeats and interfacial energy between neighboring repeats. We have been able to create consensus bacterial LRR constructs that are well-behaved, stable and unfold via cooperative transitions. By fitting a nearest neighbor Ising model to the unfolding transitions, we have determined that folding of individual repeats is unfavorable but the interactions between adjacent repeats are highly favorable, consistent with the high cooperativity observed for LRR proteins.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.urihttp://jhir.library.jhu.edu/handle/1774.2/38003
dc.languageen
dc.publisherJohns Hopkins University
dc.subjectRepeat proteinsen_US
dc.subjectfolding pathway selectionen_US
dc.subjectkineticsen_US
dc.subjectHXen_US
dc.titleDETERMINATION OF FOLDING PATHWAY SELECTION AND ORIGINS OF COOPERATIVITY OF NATURALLY OCCURRING AND DESIGNED CONSENSUS LEUCINE-RICH REPEAT PROTEINSen_US
dc.typeThesisen_US
dc.type.materialtexten_US
local.embargo.lift2015-08-01en_US
local.embargo.terms2015-08-01en_US
thesis.degree.departmentBiophysicsen_US
thesis.degree.disciplineBiophysicsen_US
thesis.degree.grantorJohns Hopkins Universityen_US
thesis.degree.grantorKrieger School of Arts and Sciencesen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePh.D.en_US
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