Membrane protein folding: beyond the two stage model

The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.     

Toward resolution of ambiguity for the unfolded state

The unfolded states in proteins and nucleic acids remain weakly understood despite their importance in folding processes; misfolding diseases (Parkinson's and Alzheimer's); natively unfolded proteins (as many as 30% of eukaryotic proteins, according to Fink); and the study of ribozymes. Research has been hindered by the inability to quantify the residual (native) structure present in an unfolded protein or nucleic acid. Here, a scaling model is proposed to quantify the molar degree of folding and the unfolded state. The model takes a global view of protein structure and can be applied to a number of analytic methods and to simulations. Three examples are given of application to small-angle scattering from pressure-induced unfolding of SNase, from acid-unfolded cytochrome c, and from folding of Azoarcus ribozyme. These examples quantitatively show three characteristic unfolded states for proteins, the statistical nature of a protein folding pathway, and the relationship between extent of folding and chain size during folding for charge-driven folding in RNA.     

Direct Observation of Parallel Folding Pathways Revealed Using a Symmetric Repeat Protein System

Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule.     

Intermediates and the folding of proteins L and G

We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted beta-1 and beta-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding, and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third beta-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally, the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first-order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.     

Routes are trees: the parsing perspective on protein folding

An important puzzle in structural biology is the question of how proteins are able to fold so quickly into their unique native structures. There is much evidence that protein folding is hierarchic. In that case, folding routes are not linear, but have a tree structure. Trees are commonly used to represent the grammatical structure of natural language sentences, and chart parsing algorithms efficiently search the space of all possible trees for a given input string. Here we show that one such method, the CKY algorithm, can be useful both for providing novel insight into the physical protein folding process, and for computational protein structure prediction. As proof of concept, we apply this algorithm to the HP lattice model of proteins. Our algorithm identifies all direct folding route trees to the native state and allows us to construct a simple model of the folding process. Despite its simplicity, our model provides an account for the fact that folding rates depend only on the topology of the native state but not on sequence composition.     

Alternate pathways for folding in the flavodoxin fold family revealed by a nucleation-growth model

A recent study of experimental results for flavodoxin-like folds suggests that proteins from this family may exhibit a similar, signature pattern of folding intermediates. We study the folding landscapes of three proteins from the flavodoxin family (CheY, apoflavodoxin, and cutinase) using a simple nucleation and growth model that accurately describes both experimental and simulation results for the transition state structure, and the structure of on-pathway and misfolded intermediates for CheY. Although the landscape features of these proteins agree in basic ways with the results of the study, the simulations exhibit a range of folding behaviours consistent with two alternate folding routes corresponding to nucleation and growth from either side of the central beta-strand.     

Toward minimalist models of larger proteins: a ubiquitin-like protein

Our recently developed off-lattice bead model capable of simulating protein structures with mixed alpha/beta content has been extended to model the folding of a ubiquitin-like protein and provides a means for examining the more complex kinetics involved in the folding of larger proteins. Using trajectories generated from constant-temperature Langevin dynamics simulations and sampling with the multiple multi-histogram method over five-order parameters, we are able to characterize the free energy landscape for folding and find evidence for folding through compact intermediates. Our model reproduces the observation that the C-terminus loop structure in ubiquitin is the last to fold in the folding process and most likely plays a spectator role in the folding kinetics. The possibility of a productive metastable intermediate along the folding pathway consisting of collapsed states with no secondary structure, and of intermediates or transition structures involving secondary structural elements occurring early in the sequence, is also supported by our model. The kinetics of folding remain multi-exponential below the folding temperature, with glass-like kinetics appearing at T/T(f) approximately 0.86. This new physicochemical model, designed to be predictive, helps validate the value of modeling protein folding at this level of detail for genomic-scale studies, and motivates further studies of other protein topologies and the impact of more complex energy functions, such as the addition of solvation forces.     

Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of alpha-lactalbumin and lysozyme

The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.     

Stepwise formation of alpha-helices during cytochrome c folding

Two models have been proposed to describe the folding pathways of proteins. The framework model assumes the initial formation of the secondary structures whereas the hydrophobic collapse model supposes their formation after the collapse of backbone structures. To differentiate between these models for real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have characterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c). We found that approximately 20% of the native helical content was organized in the first phase of folding, which is completed within milliseconds. Furthermore, we suggest the presence of a second intermediate, which has alpha-helical content resembling that of the molten globule state. Our results indicate that many of the alpha-helices are organized after collapse in the folding mechanism of cyt c.     

A circumventing role for the non-native intermediate in the folding of β-lactoglobulin

Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G ?o model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine β-lactoglobulin (βLG). This protein has an intermediate with a non-native α-helical structure, although its native form is predominantly composed of β-structure. In this study, we prepared mutants whose α-helical and β-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose β-sheet propensity was increased, showed a folding intermediate with an amount of β-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high α-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.     

Prediction of protein local structures and folding fragments based on building-block library

In recent years, protein structure prediction using local structure information has made great progress. In this study, a novel and effective method is developed to predict the local structure and the folding fragments of proteins. First, the proteins with known structures are split into fragments. Second, these fragments, represented by dihedrals, are clustered to produce the building blocks (BBs). Third, an efficient machine learning method is used to predict the local structures of proteins from sequence profiles. Finally, a bi-gram model, trained by an iterated algorithm, is introduced to simulate the interactions of these BBs. For test proteins, the building-block lattice is constructed, which contains all the folding fragments of the proteins. The local structures and the optimal fragments are then obtained by the dynamic programming algorithm. The experiment is performed on a subset of the PDB database with sequence identity less than 25%. The results show that the performance of the method is better than the method that uses only sequence information. When multiple paths are returned, the average classification accuracy of local structures is 72.27% and the average prediction accuracy of local structures is 67.72%, which is a significant improvement in comparison with previous studies. The method can predict not only the local structures but also the folding fragments of proteins. This work is helpful for the ab initio protein structure prediction and especially, the understanding of the folding process of proteins.     

Microsecond scale replica exchange molecular dynamic simulation of villin headpiece: an insight into the folding landscape

Reaching the experimental time scale of millisecond is a grand challenge for protein folding simulations. The development of advanced Molecular Dynamics techniques like Replica Exchange Molecular Dynamics (REMD) makes it possible to reach these experimental timescales. In this study, an attempt has been made to reach the multi microsecond simulation time scale by carrying out folding simulations on a three helix bundle protein, Villin, by combining REMD and Amber United Atom model. Twenty replicas having different temperatures ranging from 295 K to 390 K were simulated for 1.5 μs each. The lowest Root Mean Square Deviation (RMSD) structure of 2.5 ? was obtained with respect to native structure (PDB code 1VII), with all the helices formed. The folding population landscapes were built using segment-wise RMSD and Principal Components as reaction coordinates. These analyses suggest the two-stage folding for Villin. The combination of REMD and Amber United Atom model may be useful to understand the folding mechanism of various fast folding proteins.     

Local secondary structure content predicts folding rates for simple,two-state proteins

Many single-domain proteins exhibit two-state folding kinetics, with folding rates that span more than six orders of magnitude. A quantity of much recent interest for such proteins is their contact order, the average separation in sequence between contacting residue pairs. Numerous studies have reached the surprising conclusion that contact order is well-correlated with the logarithm of the folding rate for these small, well-characterized molecules. Here, we investigate the physico-chemical basis for this finding by asking whether contact order is actually a composite number that measures the fraction of local secondary structure in the protein; viz. turns, helices, and hairpins. To pursue this question, we calculated the secondary structure content for 24 two-state proteins and obtained coefficients that predict their folding rates. The predicted rates correlate strongly with experimentally determined rates, comparable to the correlation with contact order. Further, these predicted folding rates are correlated strongly with contact order. Our results suggest that the folding rate of two-state proteins is a function of their local secondary structure content, consistent with the hierarchic model of protein folding. Accordingly, it should be possible to utilize secondary structure prediction methods to predict folding rates from sequence alone.     

Designing cooperativity into the designed protein Top7

The topology of the designed protein Top7 is not found in natural proteins. Top7 shows signatures of non‐cooperative folding in both experimental studies and computer simulations. In particular, molecular dynamics of coarse‐grained structure‐based models of Top7 show a well‐populated C‐terminal folding‐intermediate. Since most similarly sized globular proteins are cooperative folders, the non‐natural topology of Top7 has been suggested as a reason for its non‐cooperative folding. Here, we computationally examine the folding of Top7 with the intent of making it cooperative. We find that its folding cooperativity can be increased in two ways: (a) Optimization of packing interactions in the N‐terminal half of the protein enables further folding of the C‐terminal intermediate. (b) Reduction in the packing density of the C‐terminal region destabilizes the intermediate. In practice, these strategies are implemented in our Top7 model through modifications to the contact‐map. These modifications do not alter the topology of Top7 but result in cooperative folding. Amino‐acid mutations that mimic these modifications also lead to a significant increase in folding cooperativity. Finally, we devise a method to randomize the sizes of amino‐acids within the same topology, and confirm that the structure of Top7 makes its folding sensitive to packing interactions. In contrast, the ribosomal protein S6, which has secondary structure similar to Top7, has folding which is much less sensitive to packing perturbations. Thus, it should be possible to make a sequence fold cooperatively to the structure of Top7, but to do so its side‐chain packing needs to be carefully designed. Proteins 2014; 82:364–374. © 2013 Wiley Periodicals, Inc.     

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

The folding mechanism of outer membrane proteins (OMPs) of Gram-negative bacteria into lipid bilayers has been studied using OmpA of E. coli and FomA of F. nucleatum as examples. Both, OmpA and FomA are soluble in unfolded form in urea and insert and fold into phospholipid bilayers upon strong dilution of the denaturant urea. OmpA is a structural protein and forms a small ion channel, composed of an 8-stranded transmembrane beta-barrel domain. FomA is a voltage-dependent porin, predicted to form a 14 stranded beta-barrel. Both OMPs fold into a range of model membranes of very different phospholipid compositions. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers that demonstrated a highly synchronized mechanism of secondary and tertiary structure formation of beta-barrel membrane proteins. A study on FomA folding into lipid bilayers indicated the presence of parallel folding pathways for OMPs with larger transmembrane beta-barrels.