The folding mechanism of a beta-sheet: the WW domain

The folding thermodynamics and kinetics of the Pin WW domain, a three-stranded antiparallel beta-sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature for 16 mutants, which sample all strands and turns of the molecule. The mutational phi value (Phi(m)) diagram is a smooth function of sequence, indicating a prevalence of local interactions in the transition state (TS). At 37 degrees C, the diagram has a single pronounced maximum at turn 1: the rate-limiting step during folding is the formation of loop 1. In contrast, key residues for thermodynamic stability are located in the strand hydrophobic clusters, indicating that factors contributing to protein stability and folding kinetics are not correlated. The location of the TS along the entropic reaction coordinate Phi(T), obtained by temperature-tuning the kinetics, reveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Phi(m) analysis is usually applied "perturbatively" (methyl truncation), but with Phi(T) to quantitatively assess TS shifts along a reaction coordinate, more severe mutations can be used to probe regions of the free energy surface beyond the TS.     

Effects of disulfide bonds on folding behavior and mechanism of the beta-sheet protein tendamistat

Tendamistat, a small disulfide-bonded beta-sheet protein, and its three single/double-disulfide mutants are investigated by using a modified Gō-like model, aiming to understand the folding mechanism of disulfide-bonded protein as well as the effects of removal of disulfide bond on the folding process. Our simulations show that tendamistat and its two single-disulfide mutants are all two-state folders, consistent with the experimental observations. It is found that the disulfide bonds as well as three hydrogen bonds between the N-terminal loop-0 and strand-6 are of significant importance for the folding of tendamistat. Without these interactions, their two-state behaviors become unstable and the predictions of the model are inconsistent with experiments. In addition, the effect of disulfide bonds on the folding process are studied by comparing the wild-type tendamistat and its two mutants; it is found that the removal of either of the C11-C27 or C45-C73 disulfide bond leads to a large decrease in the thermodynamical stability and loss of structure in the unfolded state, and the effect of the former is stronger than that of the later. These simulation results are in good agreement with experiments and, thus, validate our model. Based on the same model, the detailed folding pathways of the wild-type tendamistat and two mutants are studied, and the effect of disulfide bonds on the folding kinetics are discussed. The obtained results provide a detailed folding picture of these proteins and complement experimental findings. Finally, the folding nuclei predicted to be existent in this protein tendamistat as well as its mutants are firstly identified in this work. The positions of the nucleus are consistent with those argued in experimental studies. Therefore, a nucleation/growth folding mechanism that can explain the two-state folding manner is clearly characterized. Moreover, the effect by the removal of each disulfide bond on the folding thermodynamics and dynamics can also be well interpreted from their influence on the folding nucleus. The implementation of this work indicates that the modified Gō-like model really describes the folding behavior of protein tendamistat and could be used to study the folding of other disulfide-bonded proteins.     

Understanding the folding mechanism of an alpha-helical hairpin

The alpha-helical hairpin is the fundamental building block of the widespread helix-turn-helix DNA binding motif. With two antiparallel helices connected by a reverse turn, the alpha-helical hairpin structure may be regarded as a "supersecondary structural element" and, therefore, could exhibit rather unique folding properties. So far, the folding mechanism of alpha-helical hairpins has not been studied in detail and remains elusive. Herein, we examine the effects of the turn, the hydrophobic cluster, and a disulfide cross-linker on the folding kinetics of a designed alpha-helical hairpin, Z34C, using an infrared temperature-jump (T-jump) method in conjunction with site-specific mutagenesis. Our results show that Z34C folds with an ultrafast rate ( approximately 4.0 x 10(5) s(-1)) and support a folding mechanism in which the rate-limiting step corresponds to the formation of the reverse turn. On the other hand, the hydrophobic cluster and the disulfide cross-linker appear to largely stabilize the native state but not the folding transition state.     

Understanding the mechanism of beta-hairpin folding via phi-value analysis

The folding kinetics of a 16-residue beta-hairpin (trpzip4) and five mutants were studied by a laser-induced temperature-jump infrared method. Our results indicate that mutations which affect the strength of the hydrophobic cluster lead to a decrease in the thermal stability of the beta-hairpin, as a result of increased unfolding rates. For example, the W45Y mutant has a phi-value of approximately zero, implying a folding transition state in which the native contacts involving Trp45 are not yet formed. On the other hand, mutations in the turn or loop region mostly affect the folding rate. In particular, replacing Asp46 with Ala leads to a decrease in the folding rate by roughly 9 times. Accordingly, the phi-value for D46A is determined to be approximately 0.77, suggesting that this residue plays a key role in stabilizing the folding transition state. This is most likely due to the fact that the main chain and side chain of Asp46 form a characteristic hydrogen bond network with other residues in the turn region. Taken together, these results support the folding mechanism we proposed before, which suggests that the turn formation is the rate-limiting step in beta-hairpin folding and, consequently, a stronger turn-promoting sequence increases the stability of a beta-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a beta-hairpin primarily by decreasing its unfolding rate. In addition, we have examined the compactness of the thermally denatured and urea-denatured states of another 16-residue beta-hairpin, using the method of fluorescence resonance energy transfer. Our results show that the thermally denatured state of this beta-hairpin is significantly more compact than the urea-denatured state, suggesting that the very first step in beta-hairpin folding, when initiated from an extended conformation, probably corresponds to a process of hydrophobic collapse.     

Language from a biological perspective

The faculty of language is unique to the human species. This implies that there are human-specific biological changes that lie at the basis of human language. However, it is not clear what the nature of such changes are, and how they could be shaped by evolution. In this paper, emphasis is laid on describing language in a Chomskyan manner, as a mental object. This serves as a standpoint to speculate about the biological basis of the emergence and evolution of language.     

Protein folding in membranes: insights from neutron diffraction studies of a membrane beta-sheet oligomer

Studies of the assembly of the hexapeptide Acetyl-Trp-Leu₅ (AcWL₅) into β-sheets in membranes have provided insights into membrane protein folding. Yet, the exact structure of the oligomer in the lipid bilayer is unknown. Here we use neutron diffraction to study the disposition of the peptides in bilayers. We find that pairs of adjacent deuterium-labeled leucines have no well-defined peak or dip in the transmembrane distribution profiles, indicative of heterogeneity in the depth of membrane insertion. At the same time, the monomeric homolog AcWL₄ exhibits a homogeneous, well-defined, interfacial location in neutron diffraction experiments. Thus, although the bilayer location of monomeric AcWL₄ is determined by hydrophobicity matching or complementarity within the bilayer, the AcWL₅ molecules in the oligomer are positioned at different depths within the bilayer because they assemble into a staggered transmembrane β-sheet. The AcWL₅ assembly is dominated by protein-protein interactions rather than hydrophobic complementarity. These results have implications for the structure and folding of proteins in their native membrane environment and highlight the importance of the interplay between hydrophobic complementarity and protein-protein interactions in determining the structure of membrane proteins.     

Ranking the factors that contribute to protein beta-sheet folding

The formation of beta-sheet domains in proteins involves five energetically important factors: the formation of networks of hydrogen bonds and hydrophobic faces, and the residue propensities, or preferences, to be found at the edges of the beta-sheet, to adopt the extended conformation, and to make contact with other residues. These relative energy contributions define a potential energy function. Here, we show how optimizing this potential energy function reveals the formation of hydrophobic faces as the utmost factor. The potential energy function was optimized to minimize the Z-scores of the native topologies among the exhaustive sets of over 400 different beta-sheets. These results corroborate with experimental data that showed the environment of a protein is an important modulator of beta-sheet folding. The contact propensities were found to be the least important, which could explain the poor predictive power of beta-strand alignment methods based on pair-wise contact matrices.     

The mechanism of the Ni-Fe hydrogenases: a quantum chemical perspective

The catalytic cycle for the heterolytic splitting of H2 by Ni-Fe hydrogenase has been investigated in four recent quantum chemical studies. The mechanisms proposed are described and compared. Although there are clear differences in these mechanisms and in the assignments of the different states observed experimentally, there are also important points of concensus.     

Time-dependent changes in the denatured state(s) influence the folding mechanism of an all beta-sheet protein

Newt fibroblast growth factor (nFGF-1) is an approximately 15-kDa all beta-sheet protein devoid of disulfide bonds. Urea-induced equilibrium unfolding of nFGF-1, monitored by steady state fluorescence and far-UV circular dichroism spectroscopy, is cooperative with no detectable intermediate(s). Urea-induced unfolding of nFGF-1 is reversible, but the percentage of the protein recovered in the native state depends on the time of incubation of the protein in the denaturant. The yield of the protein in the native state decreases with the increase in time of incubation in the denaturant. The failure of the protein to refold to its native state is not due to trivial chemical reactions that could possibly occur upon prolonged incubation in the denaturant. (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra, limited proteolytic digestion, and fluorescence data suggest that the misfolded state(s) of nFGF-1 has structural features resembling that of the denatured state(s). GroEL, in the presence of ATP, is observed to rescue the protein from being trapped in the misfolded state(s). (1)H-(15)N HSQC data of nFGF-1, acquired in the denatured state(s) (in 8 m urea), suggest that the protein undergoes subtle time-dependent structural changes in the denaturant. To our knowledge, this report for the first time demonstrates that the commitment to adapt unproductive pathways leading to protein misfolding/aggregation occurs in the denatured state ensemble.     

Understanding the HIV coreceptor switch from a dynamical perspective

Background  

The entry of HIV into its target cells is facilitated by the prior binding to the cell surface molecule CD4 and a secondary coreceptor, mostly the chemokine receptors CCR5 or CXCR4. In early infection CCR5-using viruses (R5 viruses) are mostly dominant while a receptor switch towards CXCR4 occurs in about 50% of the infected individuals (X4 viruses) which is associated with a progression of the disease. There are many hypotheses regarding the underlying dynamics without yet a conclusive understanding.     

Engineering of betabellin 14D: disulfide-induced folding of a beta-sheet protein.

The betabellin target structure consists of 2 32-residue beta sheets packed against each other by hydrophobic interactions. We have designed, chemically synthesized, and biophysically characterized betabellin 14S, a single chain, and betabellin 14D, the disulfide-bridged double chain. The 32-residue nongenetic betabellin-14 chain (HSLTASIkaLTIHVQakTATCQVkaYTVHISE, a = D-Ala, k = D-Lys) has a palindromic pattern of polar (p), nonpolar (n), end (e), and beta-turn (t,r) residues (epnpnpnttnpnpnprrpnpnpnttnpnpnpe). Each half contains the same 14-residue palindromic pattern (underlined). Pairs of D-amino acid residues are used to favor formation of inverse-common (type-I') beta turns. In water at pH 6.5, the single chain of betabellin 14S is not folded, but the disulfide-linked betabellin 14D is folded into a stable beta-sheet structure. Thus, folding of the covalent dimer beta-bellin 14D is induced by formation of the single interchain disulfide bond. The binary pattern of alternating polar and nonpolar residues of its beta-sheets is not sufficient to induce folding. Betabellin 14D is a very water-soluble (10 mg/mL), small (64 residues), nongenetic (12 D residues) beta-sheet protein with properties (well-dispersed proton NMR resonances; Tm = 58 degrees C and delta Hm = 106 kcal/mol at pH 5.5) like those of a native protein structure.     

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Probing the kinetic cooperativity of beta-sheet folding perpendicular to the strand direction

In an attempt to determine how the folding dynamics of multistranded beta-sheets vary with the strand number, we have studied the temperature-induced relaxation kinetics of a four-stranded beta-sheet, DPDPDP. Our results show that the thermally induced relaxation of DPDPDP occurs on the nanosecond time scale; however, a comparison of the current results with those obtained on a sequence-related, three-stranded beta-sheet suggests that increasing the strand number from three to four increases the folding free energy barrier by a minimum of 0.8 kcal/mol, depending on the folding mechanism. Therefore, these results together suggest that the relaxation kinetics of DPDPDP can be analyzed according to a two-state model even though its folding may actually involve parallel (but degenerate or nearly degenerate) kinetic pathways. The apparent, two-state folding time of DPDPDP is determined to be approximately 0.44 micros at the thermal melting temperature, which makes it one of the fastest folders known to date.     

Design of beta-sheet systems for understanding the thermodynamics and kinetics of protein folding

Peptide beta-sheet systems have emerged as context-independent models for probing secondary structure propensities, the nature and magnitude of stabilizing weak interactions, and aspects of cooperativity both parallel and perpendicular to the strand direction. These systems have allowed fundamental advances in understanding non-covalent interactions relevant to both chemical and biological systems, and in describing the protein folding energy landscape.     

Understanding crown shyness from a 3-D perspective

Background and AimsCrown shyness describes the phenomenon whereby tree crowns avoid growing into each other, producing a puzzle-like pattern of complementary tree crowns in the canopy. Previous studies found that tree slenderness plays a role in the development of crown shyness. Attempts to quantify crown shyness have largely been confined to 2-D approaches. This study aimed to expand the current set of metrics for crown shyness by quantifying the characteristic of 3-D surface complementarity between trees displaying crown shyness, using LiDAR-derived tree point clouds. Subsequently, the relationship between crown surface complementarity and slenderness of trees was assessed.MethodsFourteen trees were scanned using a laser scanning device. Individual tree points clouds were extracted semi-automatically and manually corrected where needed. A metric that quantifies the surface complementarity (S_c) of a pair of protein molecules is applied to point clouds of pairs of adjacent trees. Then 3-D tree crown surfaces were generated from point clouds by computing their α shapes.Key ResultsTree pairs that were visually determined to have overlapping crowns scored significantly lower S_c values than pairs that did not overlap (n = 14, P < 0.01). Furthermore, average slenderness of pairs of trees correlated positively with their S_c score (R² = 0.484, P < 0.01), showing agreement with previous studies on crown shyness.ConclusionsThe characteristic of crown surface complementarity present in trees displaying crown shyness was succesfully quantified using a 3-D surface complementarity metric adopted from molecular biology. Crown surface complementarity showed a positive relationship to tree slenderness, similar to other metrics used for measuring crown shyness. The 3-D metric developed in this study revealed how trees adapt the shape of their crowns to those of adjacent trees and how this is linked to the slenderness of the trees.