Thermodynamics, Kinetics, and Salt dependence of Folding of YopM, a Large Leucine-rich Repeat Protein

Kloss, Ellen; Barrick, Doug

doi:10.1016/j.jmb.2008.08.069

Cited by 22 publications

(31 citation statements)

References 72 publications

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“…S1A). Cis-trans prolyl isomerization reactions are known to produce a slow, denaturant-insensitive phase during refolding, with a rate constant between 0.01 and 0.1 s −1 (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). The magnitude and denaturant dependence of the rate constant for the slow refolding phase in PP32 (triangles in Fig.…”

Section: Resultsmentioning

confidence: 99%

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

Dao

Majumdar

Barrick

2015

Proc. Natl. Acad. Sci. U.S.A.

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The leucine-rich repeat domain of PP32 is composed of five β-strand-containing repeats anchored by terminal caps. These repeats differ in sequence but are similar in structure, providing a means to connect topology, sequence, and folding pathway selection. Through kinetic studies of PP32, we find folding to be ratelimited by the formation of an on-pathway intermediate. Destabilizing core substitutions reveal a transition state ensemble that is highly polarized toward the C-terminal repeat and cap. To determine if this nucleus for folding corresponds to the most stable region of PP32, we monitored amide hydrogen exchange by NMR spectroscopy. Indeed, we find the highest protection to be biased toward the C terminus. Sequence manipulations that destabilize the C terminus spread out the transition state toward the middle of the protein. Consistent with results for helical ankyrin repeat proteins, these results suggest that local stabilities determine folding pathways.A lmost 50 y ago, Cyrus Levinthal suggested that protein folding is a guided process, rather than a random search (1). Since then, it has been proposed that rapid folding can be accomplished via a single preferred pathway (2-4). However, contrasting models of funneled, nonspecific folding capture key aspects of the folding process, including specific intermediates and transient stable structures (5, 6). To what extent does protein folding follow a single pathway, and to the extent that a single pathway dominates, what determines that pathway?For some globular proteins, specific kinetic intermediates have been identified with features of thermodynamically stable substructures of the native state. This observation suggests specific pathways that correspond to low energy folding routes (7-12). However, directly testing this thermodynamic control of folding pathway selections requires mapping of local stability. Hydrogen exchange methods have provided the most detailed energy maps of native proteins (13), but sequence-distant contacts and irregular tertiary structures of globular proteins tend to blur the boundaries of local stability.In contrast to globular proteins, elongated repeat proteins comprise tandem repeated secondary structural units, giving rise to regular topology lacking sequence-distant contacts. As a result, the contributions made by different regions of a repeat protein to its folding thermodynamics and kinetics can be easily dissected and compared. If local stability dictates folding, a preferred pathway should be observed, because repeats differ in sequence. If topology drives folding, multiple parallel pathways would be observed, because the repeats are similar in structure. For designed consensus α-helical ankyrin repeat constructs, with repeats of nearly identical sequence, folding proceeds via parallel pathways (14). In contrast, naturally occurring ankyrin repeat proteins with repeats of high sequence variation fold through preferred pathways (15-18). Regions that initiate folding correspond to low-energy structures, based on energy ...

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Section: Resultsmentioning

confidence: 99%

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

Dao

Majumdar

Barrick

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…CLRR2 displays similar cooperativity to the designed RI LRR but is much less cooperative than previously studied YopM LRR (22.6 kJ/mol M) and Internalin B LRR (10.25 kJ/mol M). 28,52,53 Binding affinity of CLLR2…”

Section: Chemical Denaturationmentioning

confidence: 99%

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide, a bacterial cell wall fragment

2014

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Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N-and C-capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.

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“…For wild-type YopM, which adheres to an equilibrium two-state mechanism, the m -value obtained for urea-induced unfolding [17] matches that predicted from chain length [14]. Since our deletion constructs are significantly shorter than the parent YopM construct, we expect the m -value to decrease linearly with the number of repeats deleted (black line, Figure 5).…”

Section: Resultsmentioning

confidence: 59%

“…To determine the effects of internal deletions on stability, urea-induced unfolding transitions were monitored by far-UV CD (a probe of secondary structure formation over the entire molecule) and tryptophan fluorescence (a probe of the environment surrounding the two tryptophans in the N-terminal α-helical cap) [17]. For all internal deletion constructs, full sigmoidal unfolding transitions were observed.…”

Section: Resultsmentioning

confidence: 99%

Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

Vieux

Barrick

2011

Biophysical Chemistry

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Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins.

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Thermodynamics, Kinetics, and Salt dependence of Folding of YopM, a Large Leucine-rich Repeat Protein

Cited by 22 publications

References 72 publications

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide, a bacterial cell wall fragment

Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

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