Folding mechanism of ketosteroid isomerase from Comamonas testosteroni

Ketosteroid isomerase (KSI) from Comamonas testosteroni is a homodimeric enzyme with 125 amino acids in each monomer catalyzing the allylic isomerization reaction at rates comparable to the diffusion limit. Kinetic analysis of KSI refolding has been carried out to understand its folding mechanism. The refolding process as monitored by fluorescence change revealed that the process consists of three steps with a unimolecular fast, a bimolecular intermediate, and most likely unimolecular slow phases. The fast refolding step might involve the formation of structured monomers with hydrophobic surfaces that seem to have a high binding capacity for the amphipathic dye 8-anilino-1-naphthalenesulfonate. During the refolding process, KSI also generated a state that can bind equilenin, a reaction intermediate analogue, at a very early stage. These observations suggest that the KSI folding might be driven by the formation of the apolar active-site cavity while exposing hydrophobic surfaces. Since the monomeric folding intermediate may contain more than 83% of the native secondary structures as revealed previously, it is nativelike taking on most of the properties of the native protein. Urea-dependence analysis of refolding revealed the existence of folding intermediates for both the intermediate and slow steps. These steps were accelerated by cyclophilin A, a prolyl isomerase, suggesting the involvement of a cis-trans isomerization as a rate-limiting step. Taken together, we suggest that KSI folds into a monomeric intermediate, which has nativelike secondary structure, an apolar active site, and exposed hydrophobic surface, followed by dimerization and prolyl isomerizations to complete the folding.     

A new method to measure cortical growth in the developing brain

Folding of the cerebral cortex is a critical phase of brain development in higher mammals but the biomechanics of folding remain incompletely understood. During folding, the growth of the cortical surface is heterogeneous and anisotropic. We developed and applied a new technique to measure spatial and directional variations in surface growth from longitudinal magnetic resonance imaging (MRI) studies of a single animal or human subject. MRI provides high resolution 3D image volumes of the brain at different stages of development. Surface representations of the cerebral cortex are obtained by segmentation of these volumes. Estimation of local surface growth between two times requires establishment of a point-to-point correspondence ("registration") between surfaces measured at those times. Here we present a novel approach for the registration of two surfaces in which an energy function is minimized by solving a partial differential equation on a spherical surface. The energy function includes a strain-energy term due to distortion and an "error energy" term due to mismatch between surface features. This algorithm, implemented with the finite element method, brings surface features into approximate alignment while minimizing deformation in regions without explicit matching criteria. The method was validated by application to three simulated test cases and applied to characterize growth of the ferret cortex during folding. Cortical surfaces were created from MRI data acquired in vivo at 14 days, 21 days, and 28 days of life. Deformation gradient and Lagrangian strain tensors describe the kinematics of growth over this interval. These quantitative results illuminate the spatial, temporal, and directional patterns of growth during cortical folding.     

Multiple routes and milestones in the folding of HIV-1 protease monomer

Proteins fold on a time scale incompatible with a mechanism of random search in conformational space thus indicating that somehow they are guided to the native state through a funneled energetic landscape. At the same time the heterogeneous kinetics suggests the existence of several different folding routes. Here we propose a scenario for the folding mechanism of the monomer of HIV-1 protease in which multiple pathways and milestone events coexist. A variety of computational approaches supports this picture. These include very long all-atom molecular dynamics simulations in explicit solvent, an analysis of the network of clusters found in multiple high-temperature unfolding simulations and a complete characterization of free-energy surfaces carried out using a structure-based potential at atomistic resolution and a combination of metadynamics and parallel tempering. Our results confirm that the monomer in solution is stable toward unfolding and show that at least two unfolding pathways exist. In our scenario, the formation of a hydrophobic core is a milestone in the folding process which must occur along all the routes that lead this protein towards its native state. Furthermore, the ensemble of folding pathways proposed here substantiates a rational drug design strategy based on inhibiting the folding of HIV-1 protease.     

Combinatorial pattern discovery approach for the folding trajectory analysis of a beta-hairpin

The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated) approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters)-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity c in RO((N + nm) log n), where N is the size of the output patterns and (n x m) is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1) The method recovers states previously obtained by visually analyzing free energy surfaces. (2) It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3) The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the choice of reaction coordinates. (An abstract version of this work was presented at the 2005 Asia Pacific Bioinformatics Conference [1].).     

A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.     

Mechanism of membrane curvature sensing by amphipathic helix containing proteins

There are several examples of membrane-associated protein domains that target curved membranes. This behavior is believed to have functional significance in a number of essential pathways, such as clathrin-mediated endocytosis, which involve dramatic membrane remodeling and require the recruitment of various cofactors at different stages of the process. This work is motivated in part by recent experiments that demonstrated that the amphipathic N-terminal helix of endophilin (H0) targets curved membranes by binding to hydrophobic lipid bilayer packing defects which increase in number with increasing membrane curvature. Here we use state-of-the-art atomistic simulation to explore the packing defect structure of curved membranes, and the effect of this structure on the folding of H0. We find that not only are packing defects increased in number with increasing membrane curvature, but also that their size distribution depends nontrivially on the curvature, falling off exponentially with a decay constant that depends on the curvature, and crucially that even on highly curved membranes defects large enough to accommodate the hydrophobic face of H0 are never observed. We furthermore find that a percolation model for the defects explains the defect size distribution, which implies that larger defects are formed by coalescence of noninteracting smaller defects. We also use the recently developed metadynamics algorithm to study in detail the effect of such defects on H0 folding. It is found that the comparatively larger defects found on a convex membrane promote H0 folding by several kcal/mol, while the smaller defects found on flat and concave membrane surfaces inhibit folding by kinetically trapping the peptide. Together, these observations suggest H0 folding is a cooperative process in which the folding peptide changes the defect structure relative to an unperturbed membrane.     

Calcium as a Crucial Cofactor for Low Density Lipoprotein Receptor Folding in the Endoplasmic Reticulum

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulse-chase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.     

Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates

Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Protein folding in vitro and in the cellular environment

The main concepts concerning protein folding have been developed from in vitro refolding studies. They state that the folding of a polypeptide chain is a spontaneous process depending only on the amino-acid sequence in a given environment. It is thermodynamically controlled and driven by the hydrophobic effect. Consequently, it has been accepted that the in vitro refolding process is a valuable model to understand the mechanisms involved during the folding of a nascent polypeptide chain in the cell. Although it does not invalidate the main rules deduced from the in vitro studies, the discovery of molecular chaperones has led to a re-evaluation of this last point. Indeed, in cells molecular chaperones are able to mediate the folding of polypeptide chains and the assembly of subunits in oligomeric proteins. The possible mechanisms by which these folding helpers act are discussed in the light of the data available in the literature. The folding process is assisted in the cell in different ways, preventing premature folding of the polypeptide chain and suppressing the incorrectly folded species and aggregates. Molecular chaperones bind to incompletely folded proteins in a conformation which suggests that the latter are in the "molten globule" state. However, very little is known about the recognition process.     

Posttransition state desolvation of the hydrophobic core of the src-SH3 protein domain

The folding thermodynamics of the src-SH3 protein domain were characterized under refolding conditions through biased fully atomic molecular dynamics simulations with explicit solvent. The calculated free energy surfaces along several reaction coordinates revealed two barriers. The first, larger barrier was identified as the transition state barrier for folding, associated with the formation of the first hydrophobic sheet of the protein. phi values calculated from structures residing at the transition state barrier agree well with experimental phi values. The microscopic information obtained from our simulations allowed us to unambiguously assign intermediate phi values as the result of multiple folding pathways. The second, smaller barrier occurs later in the folding process and is associated with the cooperative expulsion of water molecules between the hydrophobic sheets of the protein. This posttransition state desolvation barrier cannot be observed through traditional folding experiments, but is found to be critical to the correct packing of the hydrophobic core in the final stages of folding. Hydrogen exchange and NMR experiments are suggested to probe this barrier.     

Comparison of the molten globule states of thermophilic and mesophilic alpha-amylases

In recent years great interest has been generated in the process of protein folding, and the formation of intermediates during the folding process has been proven with new experimental strategies. In the present work, we have examined the molten globule state of Bacillus licheniformis alpha-amylase (BLA) by intrinsic fluorescence and circular dichroism spectra, 1-anilino naphthalene-8-sulfonate (ANS) binding and proteolytic digestion by pepsin, for comparison to its mesophilic counterpart, Bacillus amyloliquefaciens alpha-amylase (BAA). At pH 4.0, both enzymes acquire partially folded state which show characteristics of molten globule state. They unfold in such a way that their hydrophobic surfaces are exposed to a greater extent compared to the native forms. Chemical denaturation studies by guanidine hydrochloride and proteolytic digestion with pepsin show that molten globule state of BLA is more stable than from BAA. Results from gel filtration indicate that BAA has the same compactness at pH 4.0 and 7.5. However, molten globule state of BLA is less compact than its native state. The effects of polyols such as trehalose, sorbitol and glycerol on refolding of enzymes from molten globule to native state were also studied. These polyols are effective on refolding of mesophilic alpha-amylase but only slightly effect on BLA refolding. In addition, the folding pathway and stability of intermediate state of the thermophilic and the mesophilic alpha-amylases are discussed.     

Dynamics, energetics, and structure in protein folding

For many decades, protein folding experimentalists have worked with no information about the time scales of relevant protein folding motions and without methods for estimating the height of folding barriers. Protein folding experiments have been interpreted using chemical models in which the folding process is characterized as a series of equilibria between two or more distinct states that interconvert with activated kinetics. Accordingly, the information to be extracted from experiments was circumscribed to apparent equilibrium constants and relative folding rates. Recent developments are changing this situation dramatically. The combination of fast-folding experiments with the development of analytical methods more closely connected to physical theory reveals that folding barriers in native conditions range from minimally high (approximately 14RT for the very slow folder AcP) to nonexistent. While slow-folding (i.e., > or = 1 ms) single-domain proteins are expected to fold in a two-state fashion, microsecond-folding proteins should exhibit complex behavior arising from crossing marginal or negligible folding barriers. This realization opens a realm of exciting opportunities for experimentalists. The free energy surface of a protein with a marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic factors from structural factors in folding. Kinetic experiments on these proteins provide the unique opportunity to measure folding dynamics directly. Furthermore, the complex distributions of time-dependent folding behaviors expected for these proteins might be accessible to single-molecule measurements. Here, we discuss some of these recent developments in protein folding, emphasizing aspects that can serve as a guide for experimentalists interested in exploiting this new avenue of research.     

Protein folding in confined and crowded environments

Confinement and crowding are two major factors that can potentially impact protein folding in cellular environments. Theories based on considerations of excluded volumes predict disparate effects on protein folding stability for confinement and crowding: confinement can stabilize proteins by over 10k_BT but crowding has a very modest effect on stability. On the other hand, confinement and crowding are both predicted to favor conformations of the unfolded state which are compact, and consequently may increase the folding rate. These predictions are largely borne out by experimental studies of protein folding under confined and crowded conditions in the test tube. Protein folding in cellular environments is further complicated by interactions with surrounding surfaces and other factors. Concerted theoretical modeling and test-tube and in vivo experiments promise to elucidate the complexity of protein folding in cellular environments.     

Experimental determination of folding factor of benign breast cancer cell (MCF10A) and its effect on contact models and 3D manipulation of biological particles

Plasma membrane of most cells is not smooth. The surfaces of both small and large micropermeable cells are folded and corrugated which makes mammalian cells to have a larger membrane surface than the supposed ideal mode, that is, the smooth sphere of the same volume. Since cancer is an anthropic disease, cancer cells tend to have a larger membrane area than normal cells. Therefore, cancer cells have higher folding factor and larger radius than normal and healthy cells. On the other hand, the prevalence of breast cancer has prompted researchers to improve the treatment options raised for the disease in the past. In this paper, the impact of folding factor of the cell surface has been investigated. Considering that AFM is one of the most effective tools in performing the tests at micro- and nanoscales, it was used to determine the topography of MCF10 cells and then the resulting images and results were used to experimentally extract the folding factor of cells. By applying this factor in the Hertz, DMT and JKR contact models in the elastic and viscoelastic states, these models have been modified and the simulation of the three models shows that the simulation results are closer to the experimental results by considering the folding in the calculations. Additionally, the simulation of 3D manipulation has been done in both elastic and viscoelastic states with and without consideration of folding. Finally, the results were compared to investigate the effects of folding of the cell surface to the critical force and critical time of sliding and rolling in contact with the substrate and AFM tip in the 3D manipulation model.     

An entropic perspective of protein stability on surfaces

The interaction of proteins with surfaces regulates numerous processes in nature, science, and technology. In many applications, it is desirable to place proteins on surfaces in an active state, and tethering represents one manner in which to accomplish this. However, a clear understanding of how tether placement and design affects protein activity is lacking. Available theoretical models predict that proteins will be stabilized when tethered to substrates. Such models suggest that the surface reduces the number of states accessible to the unfolded state of the protein, thereby reducing the entropic cost of folding on the surface compared to the bulk case. Recent studies, however, have shown that this stabilization is not always seen. The purpose of this article is to determine the validity of the theory with a thorough thermodynamic analysis of the folding of peptides attached to surfaces. Configuration-temperature-density-of-states Monte Carlo simulations are used to examine the behavior of four different peptides of different secondary and tertiary structure. It is found that the surface does reduce the entropic cost of folding for tethered peptides, as the theory suggests. This effect, however, does not always translate into improved stability because the surface may also have a destabilizing enthalpic effect. The theory neglects this effect and assumes that the enthalpy of folding is the same on and off the surface. Both the enthalpic and entropic contributions to the stability are found to be topology- and tether-placement-specific; we show that stability cannot be predicted a priori. A detailed analysis of the folding of protein A shows how the same protein can be both stabilized and destabilized on a surface depending upon how the tethering enhances or hinders the ability of the peptide to form correct tertiary structures.     

Chaperone-like functions of high-mannose type and complex-type N-glycans and their molecular basis

It has recently become apparent that high-mannose type N-glycans directly promote protein folding, whereas complex-type ones play a crucial role in the stabilization of protein functional conformations through hydrophobic interactions with the hydrophobic protein surfaces. Here an attempt was made to understand more deeply the molecular basis of these chaperone-like functions with the aid of information obtained from spacefill models of N-glycans. The promotion of protein folding by high-mannose N-glycans seemed to be based on their unique structure, which includes a hydrophobic region similar to the cyclodextrin cavity. The promotive features of high-mannose N-glycans newly observed under various conditions furnished strong support for the view that both intra- and extramolecular high-mannose N-glycans are directly involved in the promotion of protein folding in the endoplasmic reticulum. Further, it was revealed that the N-acetyllactosamine units in complex-type N-glycans have an amphiphilic structure and greatly contribute to the formation of extensive hydrophobic surfaces and, consequently, to the N-glycan-protein hydrophobic interactions. The processing of high-mannose type N-glycans to complex-type ones seems to be an ingenious device to enable the N-glycans to perform these two chaperone-like functions.     

The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme.

We have previously proposed a hierarchical model for the folding mechanism of the Tetrahymena ribozyme that may illustrate general features of the folding pathways of large RNAs. While the role of elements in the conserved catalytic core of this ribozyme during the folding process is beginning to emerge, the participation of non-conserved peripheral extensions in the kinetic folding mechanism has not yet been addressed. We now show that the 3'-terminal P9.1-P9.2 extension of the Tetrahymena ribozyme plays an important role during the folding process and appears to guide formation of the catalytic core.