Characterization of the Heterogeneity and Specificity of Interpolypeptide Interactions in Amyloid Protofibrils by Measurement of Site-Specific Fluorescence Anisotropy Decay Kinetics

The aggregation of proteins often results in highly ordered fibrillar structures. While significant insights have been obtained on structural aspects of amyloid fibrils, little is known about the structures of protofibrils, which are presumed to be the precursors of fibrils. An understanding of the molecular mechanism of the formation of protofibrils and fibrils requires information on the landscape of interpeptide interactions. This work addresses this question by using, as a model protein, barstar, which forms protofibrils and fibrils at low (< 3) pH. Use was made of the heterogeneity of aggregate populations encountered during fibril formation. Population heterogeneity was scored through rotational dynamics monitored by time-resolved fluorescence anisotropy of an environment-sensitive fluorophore, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), attached to specific locations in the protein. Firstly, it was observed that barstar, when labeled at certain locations with 1,5-IAEDANS, did not form mixed protofibrils with the corresponding unlabeled protein. Labeled and unlabeled proteins formed protofibrils as separate populations. A two-population model of fluorescence anisotropy decay kinetics exhibiting a ‘dip-and-rise’ behavior was the main readout in arriving at this conclusion. Additional support for this conclusion came from the fluorescence lifetime of the probe 1,5-IAEDANS. Subsequently, the location of the fluorophore was moved along the length of the protein in nine mutant proteins, and the capability to form mixed fibrils was assessed. The results revealed that about two-thirds of the protein sequence at the C-terminal end of the protein was intimately involved in the formation of ordered protofibrils, probably forming the core, while the remaining one-third of the protein (i.e., the N-terminal region) remained largely noninteractive and flexible. This methodology can be used as a general strategy to identify regions of a given protein sequence involved in interprotein interactions in amyloid protofibrils.     

Determination of ultrafast protein folding rates from loop formation dynamics

Quenching of the triplet state of tryptophan by contact with cysteine can be used to measure the kinetics of loop formation in unfolded proteins. Here we show that cysteine quenching dynamics also provide a novel method for measuring folding rates when the exchange between folded and unfolded states is faster than the unquenched triplet lifetime (approximately 100 micros). We use this technique to investigate folding/unfolding kinetics of the 35 residue headpiece subdomain of the protein villin, which contains a single tryptophan residue and was engineered to contain a cysteine residue at the N terminus. At intermediate concentrations of denaturant the time-course of the triplet decay consists of two relaxations, the rates and amplitudes of which reveal the fast kinetics for folding and unfolding of this protein. The folding rates extracted using a simple kinetic model are close to those reported previously from laser-induced temperature-jump experiments that employ the change in tryptophan fluorescence as a probe. However, the results differ significantly from those reported from dynamic NMR line shape analysis on a variant with methionine at the N terminus, an issue that remains to be resolved. The analysis of the triplet quenching kinetics also shows that the quenching rates in the unfolded state increase with decreasing denaturant concentration, indicating a compaction of the unfolded protein.     

Fast Subdomain Folding Prior to the Global Refolding Transition of E. coli Adenylate Kinase: A Double Kinetics Study

The rate of folding of globular proteins depends on specific local and nonlocal intramolecular interactions. What is the relative role of these two types of interaction at the initiation of refolding? We address this question by application of a “double kinetics” method based on fast initiation of refolding of site specifically labeled protein samples and detection of the transient distributions of selected intramolecular distances by means of fast measurements of time‐resolved fluorescence resonance energy transfer. We determined the distribution of the distance between the ends of a 44‐chain segment that includes the AMP_bind domain, by labeling residues 28 and 71, in Escherichia coli adenylate kinase (AK) and the distribution of the distance between residues 18 and 203, which depends on the overall order of the molecule. That distribution shows two-state transition to the native intramolecular distance at the same rate as that of the cooperative refolding transition of the AK molecule. In sharp contrast, the distance distribution between residues 28 and 71 is already native like at the end of the dead-time of the mixing device. This fast formation of native short distance between two widely separated chain sections can be either dependent on fast folding of the AMP_bind domain or a result of a very effective nonlocal interaction between specific short clusters of hydrophobic residues. Further experiments on studying the kinetics of folding of selected structural elements in the protein will help determination of the driving force of this early folding event.     

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.     

Identification of the PXW sequence as a structural gatekeeper of the headpiece C-terminal subdomain fold

The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability.     

The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35‐residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic–aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D‐NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair‐wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic–aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.     

Multiple tryptophan probes reveal that ubiquitin folds via a late misfolded intermediate

Much of our understanding of protein folding mechanisms is derived from experiments using intrinsic fluorescence of natural or genetically inserted tryptophan (Trp) residues to monitor protein refolding and site-directed mutagenesis to determine the energetic role of amino acids in the native (N), intermediate (I) or transition (T) states. However, this strategy has limited use to study complex folding reactions because a single fluorescence probe may not detect all low-energy folding intermediates. To overcome this limitation, we suggest that protein refolding should be monitored with different solvent-exposed Trp probes. Here, we demonstrate the utility of this approach by investigating the controversial folding mechanism of ubiquitin (Ub) using Trp probes located at residue positions 1, 28, 45, 57, and 66. We first show that these Trp are structurally sensitive and minimally perturbing fluorescent probes for monitoring folding/unfolding of the protein. Using a conventional stopped-flow instrument, we show that ANS and Trp fluorescence detect two distinct transitions during the refolding of all five Trp mutants at low concentrations of denaturant: T₁, a denaturant-dependent transition and T₂, a slower transition, largely denaturant-independent. Surprisingly, some Trp mutants (Ub^M1W, Ub^S57W) display Trp fluorescence changes during T₁ that are distinct from the expected U → N transition suggesting that the denaturant-dependent refolding transition of Ub is not a U → N transition but represents the formation of a structurally distinct I-state (U → I). Alternatively, this U → I transition could be also clearly distinguished by using a combination of two Trp mutations Ub^F45W-T66W for which the two Trp probes that display fluorescence changes of opposite sign during T₁ and T₂ (Ub^F45W-T66W). Global fitting of the folding/unfolding kinetic parameters and additional folding-unfolding double-jump experiments performed on Ub^M1W, a mutant with enhanced fluorescence in the I-state, demonstrate that the I-state is stable, compact, misfolded, and on-pathway. These results illustrate how transient low-energy I-states can be characterized efficiently in complex refolding reactions using multiple Trp probes.     

N-溴代琥珀酰亚胺修饰菌紫质中色氨酸的光谱学研究

采用紫外-可见吸收光谱和荧光光谱方法研究了菌紫质（Bacteriorhodopsin,bR）中的8个色氨酸（Tryptophan,Trp）残基在被N-溴代琥珀酰亚胺（N-bromosuccinimide,NBS）修饰过程中的残基数目及对应的光谱变化。研究结果显示：随着NBS／bR摩尔比例增加逐渐被修饰的Trp残基有4个左右,如果NBS过量,则Trp残基的修饰个数最终可迭6～7个;伴随化学修饰出现Trp残基特征荧光峰值下降及峰位蓝移。研究结果揭示了bR中Trp残基可能的三种结构分布,对于进一步弄清bR中Trp-视黄醛（Retinal）偶联能量传递、单独Trp残基的荧光寿命和Tm残基在膜蛋白结构和功能中的作用具有积极而重要的意义。     

Cooperation of the prolyl isomerase and chaperone activities of the protein folding catalyst SlyD

The SlyD (sensitive to lysis D) protein of Escherichia coli is a folding enzyme with a chaperone domain and a prolyl isomerase domain of the FK506 binding protein type. Here we investigated how the two domains and their interplay are optimized for function in protein folding. Unfolded protein molecules initially form a highly dynamic complex with the chaperone domain of SlyD, and they are then transferred to the prolyl isomerase domain. The turnover number of the prolyl isomerase site is very high and guarantees that, after transfer, prolyl peptide bonds in substrate proteins are isomerized very rapidly. The Michaelis constant of catalyzed folding reflects the substrate affinity of the chaperone domain, and the turnover number is presumably determined by the rate of productive substrate transfer from the chaperone to the prolyl isomerase site and by the intrinsic propensity of the refolding protein chain to leave the active site with the native prolyl isomer. The efficiency of substrate transfer is high because dissociation from the chaperone site is very fast and because the two sites are close to each other. Protein molecules that left the prolyl isomerase site with an incorrect prolyl isomer can rapidly be re-bound by the chaperone domain because the association rate is very high as well.     

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~ 27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency > 90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

A heterodimer of human 3′-phospho-adenosine-5′-phosphosulphate (PAPS) synthases is a new sulphate activating complex

3′-Phospho-adenosine-5′-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5′-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.     

Computational design and experimental testing of the fastest-folding β-sheet protein

One of the most important and elusive goals of molecular biology is the formulation of a detailed, atomic-level understanding of the process of protein folding. Fast-folding proteins with low free-energy barriers have proved to be particularly productive objects of investigation in this context, but the design of fast-folding proteins was previously driven largely by experiment. Dramatic advances in the attainable length of molecular dynamics simulations have allowed us to characterize in atomic-level detail the folding mechanism of the fast-folding all-β WW domain FiP35. In the work reported here, we applied the biophysical insights gained from these studies to computationally design an even faster-folding variant of FiP35 containing only naturally occurring amino acids. The increased stability and high folding rate predicted by our simulations were subsequently validated by temperature-jump experiments. The experimentally measured folding time was 4.3 μs at 80 °C—about three times faster than the fastest previously known protein with β-sheet content and in good agreement with our prediction. These results provide a compelling demonstration of the potential utility of very long molecular dynamics simulations in redesigning proteins well beyond their evolved stability and folding speed.     

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca^2 + binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca^2 + is essential for stability as well as correct folding of the whole protein. In the absence of Ca^2 +, pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca^2 + addition, indicating that these are not dead-end species and that Ca^2 + drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6–7 M urea, relates to Ca^2 + dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca^2 + induced folding is not restricted to the RTX domain. Taken together, Ca^2 + binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation.     

Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops.     

The Unique Binding Mode of Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase A

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 Å resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature—a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose “whiskers” of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.     

Fixation alters fluorescence lifetime and anisotropy of cells expressing EYFP-tagged serotonin1A receptor

Fluorescence microscopic approaches represent powerful techniques to monitor molecular interactions in the cellular milieu. Measurements of fluorescence lifetime and anisotropy enjoy considerable popularity in this context. These measurements are often performed on live as well as fixed cells. We report here that formaldehyde-induced cell fixation introduces heterogeneities in the fluorescence emission of serotonin_1A receptors tagged to enhanced yellow fluorescent protein, and alters fluorescence lifetime and anisotropy significantly. To the best of our knowledge, our results constitute the first report on the effect of formaldehyde fixation on fluorescence parameters of cellular proteins. We conclude that fluorescence parameters derived from fixed cells should be interpreted with caution.     

Common Structural Transitions in Explicit-Solvent Simulations of Villin Headpiece Folding

Molecular dynamics simulations of protein folding can provide very high-resolution data on the folding process; however, due to computational challenges most studies of protein folding have been limited to small peptides, or made use of approximations such as Gō potentials or implicit solvent models. We have performed a set of molecular dynamics simulations totaling >50 μs on the villin headpiece subdomain, one of the most stable and fastest-folding naturally occurring proteins, in explicit solvent. We find that the wild-type villin headpiece reliably folds to a native conformation on timescales similar to experimentally observed folding, but that a fast folding double-norleucine mutant shows significantly more heterogeneous behavior. Along with other recent simulation studies, we note the occurrence of nonnative structures intermediates, which may yield a nativelike signal in the fluorescence measurements typically used to study villin folding. Based on the wild-type simulations, we propose alternative approaches to measure the formation of the native state.     

Optimization of a β‐sheet‐cap for long loop closure

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: R W ITVTI – loop – KKIRV W E). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: R W ITVTI‐(GGGGKK)₂GGGG‐KKIRV W E) remaining folded (ΔG_U = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: R W ITVRI W ‐(GGGGKK)₂GGGG‐ W KTIRV W E) supports a remarkably stable β‐sheet (ΔG_U = 6.3 kJ/mol at 295 K, T_m = ～55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.     

Microsecond hydrophobic collapse in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein

Using small-angle X-ray scattering combined with a continuous-flow mixing device, we monitored the microsecond compaction dynamics in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein. A significant collapse of the radius of gyration from 30 A to 23.2 A occurs within 300 micros after the initiation of refolding by a urea dilution jump. The subsequent folding after the major chain collapse occurs on a considerably longer time-scale. The protein folding trajectories constructed by comparing the development of the compactness and the secondary structure suggest that the specific hydrophobic collapse model rather than the framework model better explains the experimental observations. The folding trajectory of this alpha/beta-type protein is located between those of alpha-helical and beta-sheet proteins, suggesting that native structure determines the folding landscape.