Both turn sequence and interstrand hydrophobic side-chain-sidechain interaction have been suggested to be important determinants of ␤-hairpin stability. However, their roles in controlling the folding dynamics of ␤-hairpins have not been clearly determined. Herein, we investigated the structural stability and folding kinetics of a series of tryptophan zippers by static IR and CD spectroscopies and the IR temperature jump method. Our results support a ␤-hairpin folding mechanism wherein the rate-limiting event corresponds to the formation of the turn. We find that the logarithm of the folding rate depends linearly on the entropic change associated with the turn formation, where faster folding correlates with lower entropic cost. Moreover, a stronger turn-promoting sequence increases the stability of a ␤-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a ␤-hairpin primarily by decreasing its unfolding rate.S mall size and structural simplicity make short peptides that fold into well defined structures ideal model systems for examining factors that govern protein folding (1). Of particular interest are ␤-hairpins. With two antiparallel ␤-strands connected by a turn (or loop), the ␤-hairpin motif may be regarded as the smallest folding unit that contains tertiary contacts. Although an increasing body of evidence suggests that the ␤-hairpin can act as a folding nucleus (2-4), the mechanism by which individual ␤-hairpins fold has remained elusive. This elusiveness is partly due to the fact that so far only the folding kinetics of a few sequence-unrelated ␤-hairpins have been studied experimentally (5-9). These studies firmly demonstrated that ␤-hairpins fold on the microsecond time scale; however, the marked difference in the peptide sequence of those systems studied makes it difficult to determine explicitly the key factors that control the rate of ␤-hairpin folding.Although experimental measurements of the folding kinetics of ␤-hairpins are scarce, in the past few years a remarkable number of theoretical and computational studies have been conducted regarding the folding dynamics and energetics of a variety of ␤-hairpin systems (10-21). Results from these studies generally support the idea that the peptide sequence is an important determinant of the folding rate of ␤-hairpins. For example, the statistical model of Muñoz et al. (10) predicts that moving the hydrophobic cluster one residue closer to the turn will speed up the folding rate by 4 times, whereas the results of Thirumalai and Klimov (14,17) suggest that the turn rigidity plays a rather important role in determining the rate as well as the cooperativity of ␤-hairpin folding. Although it is still under debate whether ␤-hairpin folding begins with the formation of the turn (5, 10), interstrand hydrogen bond (16), or hydrophobic collapse (11), results from simulations generally support the idea that it involves multiple kinetic events, whereas the rate-limiting step may correspond to the assem...

We studied the microsecond folding dynamics of three ␤ hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLS aa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations. P rotein-and peptide-folding events on time scales of 1-10 s are accessible to both the fastest time-resolved experiments, such as laser temperature-jump (T-jump) spectroscopy, and to advanced simulation techniques, such as distributed computing (1-6). Combining simulation and experimental techniques in studying such systems can lead to a detailed description of folding at the molecular level, along with experimental confirmation of the predicted kinetics and thermodynamics. The ␤ hairpin, a common element in protein structures, is an important test system and a potential source of insight into the folding kinetics of larger proteins. Consequently, we have seen many inquiries into the structure and folding dynamics of ␤ hairpins in recent years (7-17). Here, we have studied Trp zippers 1-3 (TZ1-TZ3), a series of unusually stable 12-residue hairpins designed by Cochran et al. (ref. 18 and Table 1).These TZs (''TrpZips'') differ only at the turn (types IIЈ, IЈ, and D-Pro-enhanced IIЈ) and form a unique hairpin conformation in which the indole side chains from opposing pairs of Trp residues interlace to form a non-hydrogen-bonded stack or zipper along the hairpin. Our objective in this work was to explore the folding process for these peptides, as observed in hundreds of folding events simulated in atomistic molecular dynamics. To test the predicted dynamics, we compared the folding rates obtained from our simulations with experimental results from laser T-jump spectroscopy by using both Trpfluorescence and IR-absorbance probes. Materials and MethodsSimulation Methodology. Our molecular dynamics simulations used software adapted from the TINKER 3.8 (J.W. Ponder, available at http:͞͞dasher.wustl.edu͞tinker) molecular-modeling package (19). We used th...

Summary The Insulin/IGF signaling pathway (IIS) is a prominent regulator of aging of worms, flies, mice and likely humans. Delayed aging by IIS reduction protects the nematode, C. elegans, from toxicity associated with the aggregation of the Alzheimer's disease linked human peptide, Aβ. We reduced IGF signaling in Alzheimer's model mice and discovered that these animals are protected from the Alzheimer's-like disease symptoms including reduced behavioral impairment, neruoinflammation, neuronal and synpatic loss. This protection is correlated with the hyper-aggregation of Aβ leading to tightly packed, ordered plaques suggesting that one aspect of the protection conferred by reduced IGF signaling is the possible sequestration of soluble Aβ oligomers into dense aggregates of lower toxicity. These findings indicate that the IGF signaling regulated mechanism that protects from Aβ toxicity is conserved from worms to mammals and point to the modulation of this signaling pathway as a promising strategy for the development of Alzheimer's disease therapy.

A kinetic and thermodynamic survey of 35 WW domain sequences is used in combination with a model to discern the energetic requirements for the transition from two-state folding to downhill folding. The sequences used exhibit a 600-fold range of folding rates at the temperature of maximum folding rate. Very stable proteins can achieve complete downhill folding when the temperature is lowered sufficiently below the melting temperature, and then at even lower temperatures they become two-state folders again because of cold denaturation. Less stable proteins never achieve a sufficient bias to fold downhill because of the onset of cold denaturation. The model, considering both heat and cold denaturation, reveals that to achieve incipient downhill folding (barrier <3 RT) or downhill folding (no barrier), the WW domain average melting temperatures have to be >50°C for incipient downhill folding and >90°C for downhill folding.activated rate ͉ alkene peptide isosteres ͉ molecular rate ͉ speed limit ͉ stability E nergy landscape theory predicts that the entropic and enthalpic contributions to the free energy of a protein may be able to compensate for one another to the point where no significant (Ͼ3 RT) barrier appears along the folding reaction coordinate (1). Such folding is now referred to as ''type 0'' or ''downhill'' folding (1, 2). The possibility of downhill folding has been supported by a number of experiments (3-7). Kinetic measurements have focused on the transition from simple single exponential (two-state) to nonexponential (low barrier) back toward simpler (pure downhill) kinetics as the thermodynamic bias toward the native state is increased (3,6,8,9). Thermodynamic measurements with probe-dependent baselines and transition temperatures suggest that downhill folding may be possible even at the melting temperature of a protein (10-12). Recently, two engineered proteins with identical melting temperatures were compared by both kinetic and thermodynamic criteria, showing that one can be classified as a two-state folder, whereas the other can be classified as a downhill folder (13). Such results have been debated extensively in the literature (14-17).It has been suggested that the kinetics and thermodynamics of downhill folders must be very sensitive to sequence and environment because of the low barriers involved (18). Indeed, the fast folder lambda repressor has been shown to fold by either two-state, framework intermediate, or downhill mechanisms, depending on solvent conditions and sequence (6,9,13,19,20). Models for protein BBL, another fast folding protein, also indicate that it folds either in a two-state or downhill manner, depending on the exact sequence and solvent conditions (21,22).The diversity of observations suggests that criteria for downhill folding can be developed only by examining a large number of fast-folding proteins. Here, we take a survey approach to the experimental study of downhill folding. We examine a series of 35 engineered WW domains with variation in loops 1 and 2, where sequence...

Tissue-specific overexpression of the human systemic amyloid precursor transthyretin (TTR) ameliorates Alzheimer's disease (AD) phenotypes in APP23 mice. TTR-␤-amyloid (A␤) complexes have been isolated from APP23 and some human AD brains. We now show that substoichiometric concentrations of TTR tetramers suppress A␤ aggregation in vitro via an interaction between the thyroxine binding pocket of the TTR tetramer and A␤ residues 18 -21 (nuclear magnetic resonance and epitope mapping). The K D is micromolar, and the stoichiometry is Ͻ1 for the interaction (isothermal titration calorimetry). Similar experiments show that engineered monomeric TTR, the best inhibitor of A␤ fibril formation in vitro, did not bind A␤ monomers in liquid phase, suggesting that inhibition of fibrillogenesis is mediated by TTR tetramer binding to A␤ monomer and both tetramer and monomer binding of A␤ oligomers. The thousandfold greater concentration of tetramer relative to monomer in vivo makes it the likely suppressor of A␤ aggregation and disease in the APP23 mice.