Folding Path Research Articles

The final stages of folding of the membrane protein bacteriorhodopsin occur by kinetically indistinguishable parallel folding paths that are mediated by pH

The folding of the transmembrane protein bacteriorhodopsin that occurs during the binding of its retinal cofactor is investigated in a membrane-like environment. Changes in the retinal absorption band reveal two transient retinal-protein intermediate states, with apparent absorption maxima at 380 nm and 440 nm, respectively. Studies on a bacteriorhodopsin mutant of Lys216, which cannot bind retinal covalently, add to evidence that retinal is non-covalently bound in these intermediate states. The two retinal-protein intermediates are genuine intermediate states that form in parallel, each with an observed rate constant of 1.1 s−1. Meanwhile no formation of the folded state is detected. Folded bacteriorhodopsin, with all trans retinal covalently bound, forms from both retinal-bound intermediates with the same apparent rate constant of 0.0070 s−1 that is independent of retinal concentration. Retinal isomerisation then occurs with a rate constant of 0.00033 s−1 to give bacteriorhodopsin containing all trans and 13 cis-retinal. These results provide experimental evidence for multiple folding routes for a membrane protein that are pH dependent, with pH conditions determining the apparent folding route. These observed parallel folding paths are kinetically indistinguishable, which contrasts with most other observations of parallel folding pathways where only pathways with different kinetics have been reported. Furthermore, together with previous work, this study shows that bacteriorhodopsin has to populate at least two folding intermediates, during folding in the mixed lipid micelles investigated here, before the final fold is attained.

Molecular dynamics study on an adipokinetic hormone peptide in aqueous solution.

Lom-AKH-I is a member of the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family of peptides found in flying insects. A molecular dynamics simulation at room temperature (293 K) in water has been performed to survey the folding path of the Lom-AKH-I peptide in water and to establish the secondary structure of Lom-AKH-I. The obtained results indicate the presence of an undefined extended conformation.

Variations in chain compactness and topological complexity uncover folding processes in the relaxation dynamics of unfolded in vacuo lysozyme

Chain collapse and the formation of a near-native tertiary structure are believed to be two key features controlling the progress of a protein folding transition. In this work, we study the interrelation between these two properties along computer-simulated relaxation trajectories of unfolded in vacuo lysozyme. Large-scale molecular shape transitions are monitored within a space defined by two discriminating descriptors of chain compactness and entanglement (or “topological”) complexity. For the system studied here, results indicate that successful refolding into native-like conformers requires a balance between polymer collapse and a topologically “correct” organization of chain loops. Although no single factor dominates the relaxation paths, compactization appears to be a necessary condition for near-native refolding. Whenever initial collapse is limited or absent, we find a “derailed” folding path with high configurational frustration. We also show that disulfide-reduced lysozyme unfolds differently, yet relaxes to the pattern of molecular shapes characteristic of the folded states of disulfide-intact lysozyme.

PH Dependence of the Folding of Intestinal Fatty Acid Binding Protein

The folding of a mostly β-sheet protein, intestinal fatty acid binding protein, was examined over a pH range of 4 to 10 in the presence of urea. At pH values ranging from 5 to 10, folding was reversible at equilibrium by circular dichroism (CD) and fluorescence. No significant concentrations of intermediates accumulated at equilibrium, and the stability of the protein was similar over this range. However, at pH 4 and low concentrations of urea (1 to 3 M) significant time-dependent aggregation occurred. High salt concentrations increased the rate and degree of aggregation. Although higher final concentrations of urea (4 to 6 M) resolubilized these aggregates, the fluorescence and circular dichroism spectra of the protein under these conditions were not those of either the native or the unfolded protein. This state was molten globule-like, showing a more intense β-sheet CD signal and a reduced fluorescence intensity with a redshifted emission wavelength maxima compared to the native protein. Higher concentrations of urea (7 to 8 M) unfolded this molten globule form in a cooperative transition. The kinetics of unfolding and refolding were examined by stopped-flow fluorescence. The mechanism of folding and unfolding did not change over the pH range from 6 to 9, with intermediate states observed during both processes. At pH 10 additional phases were observed during both folding and unfolding. The spectral properties of these kinetic intermediates were not similar to those of the molten globule form at pH 4.0. As such, the equilibrium molten globule observed at low pH and high ionic strength does not appear to be on the folding path for this protein.

Folding mechanism of three structurally similar beta-sheet proteins.

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly beta-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, deltaGH2O) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences.

Folding mechanism of three structurally similar β‐sheet proteins

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly β-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, ΔG) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences. Proteins 33:107–118, 1998. © 1998 Wiley-Liss, Inc.

Kinetics of peptide folding: computer simulations of SYPFDV and peptide variants in water

The folding of Ser-Tyr-Pro-Phe-Asp-Val (SYPFDV), and sequence variants of this peptide (SYPYD and SYPFD) are studied computationally in an explicit water environment. An atomically detailed model of the peptide is embedded in a sphere of TIP3P water molecules and its optimal structure is computed by simulated annealing. At distances from the peptide that are beyond a few solvation shells, a continuum solvent model is employed. The simulations are performed using a mean field approach that enhances the efficiency of sampling peptide conformations. The computations predict a small number of conformations as plausible folded structures. All have a type VI turn conformation for the peptide backbone, similar to that found using NMR. However, some of the structures differ from the experimentally proposed ones in the packing of the proline ring with the aromatic residues. The second most populated structure has, in addition to a correctly folded backbone, the same hydrophobic packing as the conformation measured by NMR.Our simulations suggest a kinetic mechanism that consists of three separate stages. The time-scales associated with these stages are distinct and depend differently on temperature. Electrostatic interactions play an initial role in guiding the peptide chain to a roughly correct structure as measured by the end-to-end distance. At the same time or later the backbone torsions rearrange due to local tendency of the proline ring to form a turn: this step depends on solvation forces and is helped by loose hydrophobic interactions. In the final step, hydrophobic residues pack against each other.We also show the existence of an off the pathway intermediate, suggesting that even in the folding of a small peptide “misfolded” structures can form. The simulations clearly show that parallel folding paths are involved.Our findings suggest that the process of peptide folding shares many of the features expected for the significantly larger protein molecules.

骨組み構造の畳み込み解析

Folding structures can be a smart solution for effective transportation economizing space and for quick assemblage saving time and labor at construction sites. In the paper a numerical procedure to find one of the optimum paths to fold structures into desired configuration is proposed. Concepts of the minimum displacement path and the geometrical measurer are introduced in order to assess the validity of computed folding paths. The definition of the bifurcation point on a folding path and bifurcation point is discussed. Two illustrative examples are presented and how to compare the effectiveness of paths among different folding paths is demonstrated. Classification of bifurcation paths at a bifurcation point is attempted with the aid of group theoretic approach.

Protein folding and association: In vitro studies for self-organization and targeting in the cell

Publisher Summary The chapter discusses the protein folding and association: in vitro studies for self-organization and targeting in the cell. The chapter describes several techniques for the hierarchies of structure, stability, and folding. The chapter also discusses the mechanism of folding and association and the fundamentals of structure formation. It provides useful information for the study of folding of small single-domain proteins. In fact, after 20 years of in vitro protein folding, the practical application of denaturation/renaturation cycles in the downstream processing of recombinant proteins forced researchers in the field to ask questions that focused on the cellular aspects of folding. In case of the chaperones, the fundamental importance, beyond the stress response, has been clearly shown: (i) for the kinetic partioning among folding, association, and aggregation of oligomeric proteins; (ii) for protein targeting, e.g., translocation of mitochondrial proteins across membranes; and (iii) for processes involved in morphogenesis, such as growth and self-assembly of phages. The elucidation of the detailed folding mechanism of a given protein would require the complete description of the nascent (unfolded) and final native states, together with all the intermediates along the multistep folding path.

Thermodynamic peptide folding path proposed

If scientists could watch a protein fold from a chain of amino acids, perhaps it would look like a film run backward: a loose, flailing strand begins to kink and coil, seemingly defying entropy as it assembles into a precise, compact structure. What sends a protein down this complex and still-mysterious path, it is believed, are fluctuating attractions in small, localized sections of the chain, which fold into short helices or sheets in preparation for global collapse into the final three-dimensional structure. For this reason, some biochemists find the behavior of short peptides particularly important. Recently, a University of California, Santa Cruz, lab led by chemistry professor Glenn L. Millhauser found some new twists in the character of such chains as they form helices. Using electron spin resonance (ESR), the scientists found that rather than forming the very common α helix, as previously thought, a group of small peptides may actually contain substantial portions of ...

The substructural organization of the chromosome core (scaffold) in meiotic chromosomes of Trilophidia annulata

SummaryThe substructural organization of chromosome cores or nonhistone proteins was studied within intact metaphase chromosomes at the second meiotic division in the grasshopper Trilophidia annulata by silver staining as well as light microscopy and whole mount electron microscopy of squash chromosomes. Our results revealed that the metaphase II chromosome contains a longitudinal, helical coiling core structure. Probably the two last organizational levels of the core packaging are achieved by helical coiling. The core structure retains the morphological characteristics of the original metaphase chromosome, surrounded by a halo of dispersed materials, which may be composed mainly of nonhistone proteins. The kinetochore is found to be connected with the chromosome core. The present findings combined with our previous observations on the helical structure of metaphase II chromosomes suggest that the folding path of the internal core structure in metaphase chromosomes is consistent with the final helical arrangement of the chromosome itself. These observations also imply that in condensed metaphase chromosomes nonhistone protein may form a compact network structure with helical appearance, which extends throughout the entire chromosome.

Ruling folding path from a look at protein structures

Ruling folding path from a look at protein structures

Metaphase chromosome structure: Bands arise from a differential folding path of the highly AT-rich scaffold

Using the highly AT-specific fluorchrome daunomycin, a longitudinal optical signal called AT queue, thought to arise from a line-up of the highly AT-rich scaffold-associated regions (SARs) by the scaffolding, was identified in native chromosomes. Fluorescence banding is proposed to result from a differential folding path of the AT queue during its progression from telomere to telomere. The AT queue is tightly coiled or folded in a Q band, the resulting transverse striations across the chromatid, which also represent Giemsa subbands, generating a bright AT-rich signal over the Q region. The R bands, in contrast, contain a more central (unfolded) AT queue, yielding an AT-dull signal over the R regions. The AT queue is identified by immunofluorescence against topoisomerase II (topo II) and HMG-I Y as the scaffold of native chromosomes; the fluorescence signal from both proteins is akin to a detailed Q-type banding pattern. Native chromosomes appear assembled according to the loop-scaffold model.

FOLLOWING PATHS OF SYMMETRY-BREAKING BIFURCATION POINTS

We propose a pseudo-arclength continuation algorithm for computing paths of Z 2-symmetry-breaking bifurcation points for two-parameter nonlinear elliptic problems. The algorithm consists of an Euler predictor step and a solution step composed of a sequence of Newton iterations. This work generalizes the algorithm of Werner and Spence for locating a one-parameter symmetry-breaking bifurcation point by using the approach of Keller and Fier for following a (two-parameter) path of limit points (a "fold"). By repeated use of the bordering algorithm, we solve linear systems whose matrix is the "symmetric" Jacobian or "antisymmetric" Jacobian, thus fully exploiting any (block tridiagonal) structures present. We give numerical results for the steady, axisymmetric flow between rotating coaxial cylinders (Taylor–Couette flow). For finite cylinders, we compute the fold curve and path of symmetry-breaking bifurcation points for small aspect ratios, and illustrate a new method to accurately locale the two Z 2-symmetric codimension one singularities. For infinite cylinders, we show the projections on the (aspect ratio, Reynolds number) plane of the folds and bifurcation point paths in the neighborhood of the two-cell/four-cell neutral curve crossing. We numerically verify a conjecture of Meyer–Spasche and Wagner concerning the connection of two neutral-curve crossings by a path of secondary subharmonic bifurcations.

Noninteracting local-structure model of folding and unfolding transition in globular proteins. I. Formulation.

AbstractA statistical‐mechanical model (a noninteracting local structure model) of folding and unfolding transition in globular proteins is described and a formulation is given to calculate the partition function. The process of transition is discussed in this model within the framework of equilibrium statistical mechanics. In order to clarify the range of applicability of such an approach, the characteristics of the folding and unfolding transition in globular proteins are analyzed from the statistical‐physical point of view. A theoretical advantage is pointed out in studying folding and unfolding processes taking place as conformational fluctuations in individual protein molecules under macroscopic equilibrium at the melting temperature. In this case, paths of folding and unfolding are shown to be identical in the statistical sense. A key to the noninteracting local structure model lies in the concept of local structures and the assumption of the absence of interactions between local structures. A local structure is defined as a continuous section of the chain which takes the same or similar local conformation as in the native conformation. The assumption of the absence of inter‐actions between local structures endows the model with the remarkable character that its partition function can be calculated exactly; thereby the equilibrium population of various conformations along the folding and unfolding paths can be discussed only by a knowledge of the folded native conformation.