Molecular picture of folding of a small α/β protein

124

Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of singlechain monellin composed of a five-stranded ␤-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 s after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of ␤-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064 -16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.energy landscape ͉ protein folding ͉ submillisecond dynamics ͉ x-ray scattering C haracterization of the folding dynamics for proteins and their free energy landscapes offers a key to understanding protein folding phenomena (1, 2). The coarse-grained approximation of intraprotein interactions (a ''funneled'' landscape) has been proposed (3, 4) and has succeeded in explaining the structures of proteins in the folding transition state (5-8). However, the molecular basis of the funneled landscape, which is realized by a complex interplay of various intraprotein interactions and polypeptide dynamics, is largely unknown. For example, although the hydrophobic interaction and hydrogen bond (H-bond) are the major stabilizing interactions for the specific folded conformations of proteins, their contribution to the energy landscape, including the unfolded conformations, is controversial (9). Furthermore, the relationship between protein folds and the characteristics of landscapes has not been well investigated. Therefore, the observation of the entire folding process of proteins with various folds is required to characterize the free energy landscapes.We previously observed the folding dynamics of ␣-helical proteins, cytochrome c (cyt c) and apomyoglobin (apoMb), based on helical content and compactness, which mainly reflect the formation of the H-bonds and hydrophobic interactions, respectively (10-12). The characterized landscapes of these proteins demonstrated the cooperative acquisition of the helical structure and compactness after the initial collapse and suggested that the hydrophobic environment created by the collapse facilitates the subsequent conformational search. ...

Section: L-shaped Conformational Landscape Of Smnsupporting

confidence: 82%

supporting

confidence: 80%

Specific collapse followed by slow hydrogen-bond formation of β-sheet in the folding of single-chain monellin

Kimura

Uzawa

Ishimori

et al. 2005

124

“…In protein L, the N-terminal hairpin is predominantly formed, whereas the C-terminal hairpin is unformed (25,26); however, in protein G, the C-terminal hairpin has been shown to form ahead of the N-terminal hairpin (27)(28)(29)(30). These differences emerged in the same simple model used here, which subsequently led to an explanation in terms of the enthalpy and entropy differences associated with the formation of each hairpin (24).…”

mentioning

confidence: 83%

The structural basis for biphasic kinetics in the folding of the WW domain from a formin-binding protein: Lessons for protein design?

Karanicolas

Brooks

2003

Self Cite

124

156

The mechanism of formation of ␤-sheets is of great importance because of the significant role of such structures in the initiation and propagation of amyloid diseases. In this study we examine the folding of a series of three-stranded antiparallel ␤-sheets known as WW domains. Whereas other WW domains have been shown to fold with single-exponential kinetics, the WW domain from murine formin-binding protein 28 has recently been shown to fold with biphasic kinetics. By using a combination of kinetics and thermodynamics to characterize a simple model for this protein, the origins of the biphasic kinetics is found to lie in the fact that most of the protein is able to fold without requiring one of the ␤-hairpins to be correctly registered. The correct register of this hairpin is enforced by a surface-exposed hydrophobic contact, which is not present in other WW domains. This finding suggests the use of judiciously chosen surface-exposed hydrophobic pairs as a protein design strategy for enforcing the desired strand registry.␤-sheet ͉ ␤-strand ͉ negative design ͉ strand register A n understanding of the mechanism by which proteins reach their particular native state from the vast number of unfolded conformations will have broad-reaching implications, ranging from the prediction of protein structure from sequence to understanding diseases that originate from protein misfolding. Small model peptides and proteins have lent invaluable insight into the protein-folding process, as they afford simple systems in which the general features of folding may be elucidated (1, 2).Because of the local nature of the interactions, the formation of helices has proven considerably easier to understand using simple models (3-5) than has the formation of ␤-sheets. Accordingly, the principles that govern the formation of ␤-sheets are not well understood, despite the fact that the formation of intermolecular ␤-sheets is thought to be the crucial event in the initiation and propagation of amyloid diseases such as Alzheimer's disease (6) and spongiform encephalopathy (7).To further an understanding of the elements responsible for stability in ␤-sheets, de novo design methods have, in two cases, been used to construct three-stranded antiparallel ␤-sheets (8, 9), which have subsequently become the subject of both theoretical (10-12) and experimental (13, 14) folding studies. To ensure the generality of results toward natural proteins, however, we opt to study a series of three-stranded antiparallel ␤-sheet domains found in a variety of proteins: the WW domains (Fig. 1a).WW domains, which bind proline-rich peptide sequences, have been identified in Ͼ200 nonredundant proteins to date (15). Because of the attractiveness of WW domains as a model for ␤-sheet formation, they have been the focus of several previous folding studies. The initial study of these systems examined the thermodynamics and kinetics of folding of the human Yes-associated protein (hYAP) WW domain (16). A subsequent study made use of the more stable Pin WW domain, to characteriz...

“…Finally, we can investigate the interplay between protein ordering and dehydration of the protein core; this question has been addressed in several recent papers (36)(37)(38)(39)(40), and it is now quantified so that a unified view for the folding mechanism becomes possible. The average number of water molecules coordinated to the backbone carbonyl oxygen atoms provides a measure of the degree of protein hydration.…”

Section: Resultsmentioning

confidence: 99%

Folding a protein in a computer: An atomic description of the folding/unfolding of protein A

Garcı́a

Onuchic

2003

322

334

We study the folding mechanism of a three-helix bundle protein at atomic resolution, including effects of explicit water. Using replica exchange molecular dynamics we perform enough sampling over a wide range of temperatures to obtain the free energy, entropy, and enthalpy surfaces as a function of structural reaction coordinates. Simulations were started from different configurations covering the folded and unfolded states. Because many transitions between all minima at the free energy surface are observed, a quantitative determination of the free energy barriers and the ensemble of configurations associated with them is now possible. The kinetic bottlenecks for folding can be determined from the thermal ensembles of structures on the free energy barriers, provided the kinetically determined transition-state ensembles are similar to those determined from free energy barriers. A mechanism incorporating the interplay among backbone ordering, sidechain packing, and desolvation arises from these calculations. Large ⌽ values arise not only from native contacts, which mostly form at the transition state, but also from contacts already present in the unfolded state that are partially destroyed at the transition. T he energy landscape theory and the funnel concept, along with a new generation of experiments, have created a ''new view'' of protein folding (1-4). Folding is best described as an ensemble of converging pathways toward the native structure. For good folding sequences, these paths are all energetically very similar with barriers between them on the k B T energy scale. Minor fluctuations due to environmental or mutational changes may vary the path probabilities, but the overall global landscape flow will be only weakly altered. This leads to a key result of the energy landscape theory: protein folding dynamics can be properly understood as the diffusion of an ensemble of protein configurations over a low-dimensional free energy surface, which may be constructed by using many different order parameters.In this article, we describe the free energy landscape for protein folding simulated with full atomic details of both protein and solvent, over a broad range of temperatures. For our studies, a natural choice is the 10-55 helical fragment B of protein A from Staphylococcus aureus, using the replica exchange molecular dynamics (REMD) algorithm described by Sugita and Okamoto (5). Protein A folds into a simple three-helix bundle (6) whose folding has been widely studied by using minimalist and all-atom simulations (7-17) as well as experiments (18,19). All of these different studies provided some information about the folding mechanism and the nature of the transition-state ensemble (TSE). The study presented here aims to establish a quantitative picture by integrating the earlier qualitative findings with detailed simulations. We simulated 82 replicas of the water-protein system, with temperature, T, ranging from 277 to 548 K, and each replica was started from a different configuration spanning the space of folde...