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

Repeat proteins;folding pathway selection;kinetics;HX;Biophysics

Dao, Thuy PhuongGarcia-Moreno, Bertrand E. ;

Johns Hopkins University

关键词: Repeat proteins;    folding pathway selection;    kinetics;    HX;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/38003/DAO-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Thermodynamic 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.