Fine Structure Analysis of a Protein Folding Transition State; Distinguishing Between Hydrophobic Stabilization and Specific Packing

Anil, Burcu; Sato, Satoshi; Cho, Jae-Hyun; Raleigh, Daniel P.

doi:10.1016/j.jmb.2005.08.054

Cited by 50 publications

(65 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the S-peptide/S-protein system this type of replacement would also lead to classical ϕ-values at most positions, due to decreased hydrophobicity (see Table 2). A study on the effect of hydrophobicity on the folding kinetics of the small monomeric protein NTL9 also revealed that increased hydrophobicity can accelerate folding despite a destabilization of the native state (36). Together with our results these observations suggest that loosely packed hydrophobic interactions are sufficient to position the different parts of the polypeptide chain correctly relative to each other in the transition state for protein folding and for protein-protein interactions.…”

Section: Role Of Hydrophobic Interactions In Protein Folding Transitisupporting

confidence: 80%

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

Bachmann

Wildemann

Praetorius

et al. 2011

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

116

147

View full text Add to dashboard Cite

Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association.phi-value analysis | protein-protein interaction | encounter complex formation | thioxo peptide bond C onformational changes in proteins play an important role in many biological processes. A detailed understanding of the mechanism of conformational transitions requires knowledge of the structural and dynamic properties of the initial and final states as well as the characterization of the transition barrier separating them. Site-directed mutagenesis proved a powerful tool to determine the properties of transition states for reactions involving proteins. Comparing the effect of amino acid replacements on kinetics and thermodynamics of a reaction identifies the interactions that are important for the rate-limiting step of a process. This method has been successfully applied to characterize transition states for protein folding (1, 2), for proteinprotein interactions (3-5) and for conformational changes in folded proteins (6). Protein folding transition states were shown to have native-like topology in the whole protein or in major parts of the structure (7-9) and it is commonly assumed that this includes the presence of native-like secondary structure (10, 11). Because site-directed mutagenesis can only modify amino acid side chains, it is still unclear at which stage of a folding reaction backbone hydrogen bonds in secondary structure elements are formed. Several approaches have been applied to test for secondary structure in protein folding transition states. Replacing amino acids by glycyl and prolyl residues leads to changes in backbone conformation, which was used to probe α-helix formation (12), but these replacements introduce additional effects due to altered side-chain interactions. Introducing ester bonds into the polypeptide backbone (13) also has multiple effects because it leads to the loss of a backbone hydrogen bonding donor and strongly increases conformational flexibility of the polypeptide backbone. Deuteration of a...

show abstract

Section: Role Of Hydrophobic Interactions In Protein Folding Transitisupporting

confidence: 80%

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

Bachmann

Wildemann

Praetorius

et al. 2011

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

116

147

View full text Add to dashboard Cite

show abstract

“…8 It seems entirely reasonable that the position of the transition state of NTL9 is sensitive to temperature since it is known that formation of hydrogen bonds and a loosely packed hydrophobic core is more important than formation of specific side-chain interactions for the folding of this protein. 27,35 Thus, a perturbation such as temperature that globally effects these interactions, instead of local perturbations such as site-specific mutations, appears to be a particularly good method for detecting transition-state movement.…”

Section: Discussionmentioning

confidence: 99%

Temperature-Dependent Hammond Behavior in a Protein-Folding Reaction: Analysis of Transition-State Movement and Ground-State Effects

Taskent

Cho

Raleigh

2008

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed alpha-beta protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 degrees C. Plots of the log of the observed first-order rate constant versus denaturant concentration, "chevron plots," displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford beta parameter, beta(T), shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of Delta G versus Delta G degrees, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.

show abstract

“…Similarly, Davidson examined the effect of substitution of nine core residues of the SH3 domain (Northey et al 2002), and Raleigh substituted six core residues of the NTL9 domain (Anil et al 2005). In both studies, nonpolar (or aromatic) residues were substituted with residues that were designed to change either the local hydrophobicity or the contacting surfaces, and the rate of refolding was measured.…”

Section: O'neill Et Al (O'neill and Robert Matthews 2000)mentioning

confidence: 99%

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

et al. 2013

View full text Add to dashboard Cite

The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a "bottom-up" mechanism. Nonlocal interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an "up-down" mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the "loop hypothesis", is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the timeresolved FRET-based "double kinetics" method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.

show abstract

Fine Structure Analysis of a Protein Folding Transition State; Distinguishing Between Hydrophobic Stabilization and Specific Packing

Cited by 50 publications

References 38 publications

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

Temperature-Dependent Hammond Behavior in a Protein-Folding Reaction: Analysis of Transition-State Movement and Ground-State Effects

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

Contact Info

Product

Resources

About