Ultrafast folding of WW domains without structured aromatic clusters in the denatured state

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...

Section: Discussionmentioning

confidence: 99%

“…Later, the thermodynamics and kinetics of folding were compared between the following three WW domains (18): one from hYAP, one from murine forminbinding protein (FBP) 28, and a de novo-designed WW domain. All three of the aforementioned studies (16)(17)(18) reported singleexponential kinetics for folding, corresponding to an apparent two-state mechanism.…”

mentioning

confidence: 99%

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

124

156

“…β-sheet formation. It should be noted that the WW domains have been the subject of extensive theoretical (1,14,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) and experimental (15)(16)(17)(29)(30)(31)(32)(33)(34)) studies because of their small size, biological importance (35), and interesting fast-folding kinetics.…”

Section: Significancementioning

confidence: 99%

Folding kinetics of WW domains with the united residue force field for bridging microscopic motions and experimental measurements

Zhou

Maisuradze

Suñol

et al. 2014

To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecondtimescale canonical Langevin molecular dynamics simulations of the triple β-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single-and double-exponential kinetics and their correlations with two-and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding.FBP28 WW domain | nonnatural variants | folding rates | free-energy landscapes | millisecond-timescale canonical MD simulations R ecent advances in computer simulation techniques have facilitated the direct study of the folding process of small fastfolding proteins, using all-atom force fields (1). However, it is important to validate the simulation methodologies, and the only way to accomplish this is a quantitative comparison with experimental data with proper statistics. The validation of all-atom simulation methodologies is still a major problem because of the differences between the experimental timescale (from multiple microseconds to seconds) and the theoretical one (from hundreds of nanoseconds to microseconds). To overcome this problem, many approximate coarse-grained methods have been developed during the past decade (2-5). One of them makes use of a physics-based united-residue (UNRES) force field developed in our group over the past years (6-14) (SI Appendix, Fig. S1 and SI Materials and Methods).The folding and unfolding rates are among the most accessible quantitative observables for two-and multistate folding proteins; therefore, a study of protein folding kinetics can bridge microscopic motions and the world of experimental measurements. In analyzing protein folding kinetics, the differential rate equations and their integrated forms become more complex as the num...

“…1N), has been shown to fold with biphasic kinetics exhibiting intermediates during folding (3,5,6,(11)(12)(13)(14)(15)(16). We address this problem here with the design of new FBP28 WW domain mutants and by examining their structural properties and folding kinetics.Because of the small size, fast folding kinetics, and biological importance, the formation of intermolecular β-sheets is thought to be a crucial event in the initiation and propagation of amyloid diseases, such as Alzheimer's disease, and spongiform encephalopathy, FBP28, and other WW domain proteins (e.g., Pin1 and FiP35) have been the subjects of extensive experimental (4,11,(17)(18)(19)(20)(21)(22)(23) and theoretical (3,5,6,(12)(13)(14)(15)(16)(24)(25)(26)(27) studies. However, a folding mechanism of the FBP28 was debatable for a long time because of its complexity.…”

mentioning

confidence: 99%

Preventing fibril formation of a protein by selective mutation

Maisuradze

Medina

Kachlishvili

et al. 2015

The origins of formation of an intermediate state involved in amyloid formation and ways to prevent it are illustrated with the example of the Formin binding protein 28 (FBP28) WW domain, which folds with biphasic kinetics. Molecular dynamics of protein folding trajectories are used to examine local and global motions and the time dependence of formation of contacts between C α s and C β s of selected pairs of residues. Focus is placed on the WT FBP28 WW domain and its six mutants (L26D, L26E, L26W, E27Y, T29D, and T29Y), which have structures that are determined by high-resolution NMR spectroscopy. The origins of formation of an intermediate state are elucidated, viz. as formation of hairpin 1 by a hydrophobic collapse mechanism causing significant delay of formation of both hairpins, especially hairpin 2, which facilitates the emergence of an intermediate state. It seems that three-state folding is a major folding scenario for all six mutants and WT. Additionally, two-state and downhill folding scenarios were identified in ∼15% of the folding trajectories for L26D and L26W, in which both hairpins are formed by the Matheson-Scheraga mechanism much faster than in three-state folding. These results indicate that formation of hairpins connecting two antiparallel β-strands determines overall folding. The correlations between the local and global motions identified for all folding trajectories lead to the identification of the residues making the main contributions in the formation of the intermediate state. The presented findings may provide an understanding of protein folding intermediates in general and lead to a procedure for their prevention.A n intermediate state in protein folding is involved in amyloid fibril formation, which is responsible for a number of neurodegenerative diseases (1-7). Therefore, prevention of the aggregation of folding intermediates is one of the most important problems to surmount. Hence, it is necessary to determine the mechanism by which an intermediate state is formed. For example, one of the members of the WW domain family (8, 9), the triple β-stranded WW domain from the Formin binding protein 28 (FBP28; Protein Data Bank ID code 1E0L) (10) (Fig. 1N), has been shown to fold with biphasic kinetics exhibiting intermediates during folding (3,5,6,(11)(12)(13)(14)(15)(16). We address this problem here with the design of new FBP28 WW domain mutants and by examining their structural properties and folding kinetics.Because of the small size, fast folding kinetics, and biological importance, the formation of intermolecular β-sheets is thought to be a crucial event in the initiation and propagation of amyloid diseases, such as Alzheimer's disease, and spongiform encephalopathy, FBP28, and other WW domain proteins (e.g., Pin1 and FiP35) have been the subjects of extensive experimental (4,11,(17)(18)(19)(20)(21)(22)(23) and theoretical (3,5,6,(12)(13)(14)(15)(16)(24)(25)(26)(27) studies. However, a folding mechanism of the FBP28 was debatable for a long time because of its complexity. There ar...