A high-resolution probe of protein folding

Protein folding is a central problem in the biological sciences. To generate residue-specific information on the equilibrium folding of cytochrome c, we have semisynthesized the protein with specifically deuterated residues. The C-D bonds may be easily visualized in an otherwise transparent region of the IR spectra, even at high protein and denaturant concentrations. Plotted as a function of added guanidine hydrochloride denaturant, the absorption intensities reveal that the protein undergoes a conformational change at the protein-based ligand, Met80, which is then followed by a more global unfolding at 2.3 M denaturant. Deuteration and characterization of other residues in cytochrome c, or other protein of interest, should provide complete views of folding with residue specific detail that is capable of resolving even the most rapidly interconverting intermediates.     

A small-molecule probe for hepatitis C virus replication that blocks protein folding

The hepatitis C virus (HCV) is a growing global health problem. Small molecules that interfere with host-viral interactions can serve as powerful tools for elucidating the molecular mechanisms of pathogenesis and defining new strategies for therapeutic development. Using a cell-based screen involving subgenomic HCV replicons, we identified the ability of 18 different abscisic acid (ABA) analogs, originally developed as plant growth regulators, to inhibit HCV replication. Three of these were further studied. One compound, here named origamicin, showed antiviral activity through the inhibition of host proteins involved in protein folding. Origamicin could therefore be an important tool for studying the maturation of both host and viral proteins. Herein we demonstrate an application for molecular scaffolds based on ABA for mammalian cell targets involved in protein folding.     

Molecular folding screen: folding and unfolding of 1,8-anthrylene-ethynylene oligomers by photochemical cycloaddition and thermal cycloreversion

Molecular folding screens consisting of anthracene plates and acetylene linkers stereoselectively fold into a zigzag form by [4 + 4]photocycloaddition, and unfold by thermal cycloreversion.     

Two-dimensional fluorescence resonance energy transfer as a probe for protein folding: a theoretical study

We describe a two-dimensional (2D), four-color fluorescence resonance energy transfer (FRET) scheme, in which the conformational dynamics of a protein is followed by simultaneously observing the FRET signal from two different donor-acceptor pairs. For a general class of models that assume Markovian conformational dynamics, we relate the properties of the emission correlation functions to the rates of elementary kinetic steps in the model. We further use a toy folding model that treats proteins as chains with breakable cross-links to examine the relationship between the cooperativity of folding and FRET data and to establish what additional information about the folding dynamics can be gleaned from 2D, as opposed to one-dimensional FRET experiments. We finally discuss the potential advantages of the four-color FRET over the three-color FRET technique.     

Three-pulse photon echo peak shift spectroscopy as a probe of flexibility and conformational heterogeneity in protein folding

We investigate the equilibrium unfolding of Zn-cytochrome c in guanidine hydrochloride by three-pulse photon echo peak shift (3PEPS) spectroscopy. Unexpectedly, the measurements reveal that inhomogeneous broadening of the sample at the midpoint of the denaturation is larger than that of either native or unfolded states. To interpret this finding, we present simulations of the peak shift for both two-state and three-state unfolding models. Both the denaturant concentration dependence of the asymptotic peak shift (APS) and the wavelength dependence of the APS at the midpoint of the denaturation are different for the two models. Our data are consistent with two-state unfolding.     

Thermal and photochemical oxidation of 2-acetylcyclopentanone with atmospheric oxygen

The major products of thermal (acetone, CaCl₂ excess, reflux) and photochemical (acetone or CCl₄, room temperature) oxidation of 2-acetylcyclopentanone with atmospheric oxygen are 2-acetyl-2-hydroxycyclopentanone, 2-acetyl-2-hydroxymethylcyclopentanone, 1,1′-diacetyl-1,1′-bicyclopentyl-2,2′-dione, 2-acetoxycyclopentanone, 5,6-dioxoheptanoic acid, glutaric acid, and glutaric anhydride. The formation of 2-acetyl-2-hydroxycyclopentanone is the first example of the direct α-hydroxylation of β-dicarbonyl compound under the conditions described.     

Temperature effects on the nucleation mechanism of protein folding and on the barrierless thermal denaturation of a native protein

Recently, the authors proposed a kinetic model for the nucleation mechanism of protein folding where a protein was treated as a heteropolymer with all the bonds and bond angles equal and constant. As a crucial idea of the model, an overall potential around a cluster of native residues in which a protein residue performs a chaotic motion is considered to be a combination of three potentials: effective pairwise, average dihedral, and confining. The overall potential as a function of the distance from the cluster center has a double well shape which allows one to determine the rates with which the cluster emits and absorbs residues by using a first passage time analysis. One can then develop a kinetic theory for the nucleation mechanism of protein folding and evaluate the protein folding time. In the present paper we evaluate the optimal temperature at which the protein folding time is the shortest. A method is also proposed to determine the temperature dependence of the folding time without carrying out the time consuming calculations for a series of temperatures. Using Taylor series expansions in the formalism of the first passage time analysis, one can calculate the temperature dependence of the cluster emission and absorption rates in the vicinity of some temperature T(0) if they are known at T(0). Thus one can evaluate the protein folding time t(f) at any other temperature T in the vicinity of T(0) at which the folding time t(f) is known. We also present a model for the thermal denaturation of a protein occurring via the decay of the native structure of the protein. Due to a sufficiently large temperature increase or decrease, the rate with which a cluster of native residues within a protein emits residues becomes larger than the absorption rate in the whole range of cluster sizes up to the size of the whole protein. This leads to the unfolding of the protein in a barrierless way, i.e., as spinodal decomposition. Knowing the cluster emission and absorption rates as functions of temperature and cluster size, one can find the threshold temperatures of cold and hot barrierless denaturation as well as the corresponding unfolding times. Both proposed methods are illustrated by numerical calculations for two model proteins, one consisting of 124 amino acids, the other consisting of 2500 residues. The first one roughly mimicks a bovine pancreatic ribonuclease while the second one is a representative of the largest proteins which are extremely difficult to study by straightforward Monte Carlo or molecular dynamics simulations.     

Phosphorescence from nylon polymers: Effects of thermal and photochemical oxidation

The phosphorescence from the nylon polymers 6, 66, 11, and 12 has been studied from the aspects of excitation and emission wavelength location and of emission lifetime. In contrast to the other nylons, the 66 polymer exhibits two distinct phosphorescence bands, both of which are sensitive to thermal and photochemical oxidation of the polymer. The species responsible for these emissions are concluded to be carbonyl in nature and their role in the complex reactions that occur during thermal and photochemical oxidation of the polymer is discussed.     

Frustration and hydrophobicity interplay in protein folding and protein evolution

A lattice model is used to study mutations and compacting effects on protein folding rates and folding temperature. In the context of protein evolution, we address the question regarding the best scenario for a polypeptide chain to fold: either a fast nonspecific collapse followed by a slow rearrangement to form the native structure or a specific collapse from the unfolded state with the simultaneous formation of the native state. This question is investigated for optimized sequences, whose native state has no frustrated contacts between monomers, and also for mutated sequences, whose native state has some degree of frustration. It is found that the best scenario for folding may depend on the amount of frustration of the native structure. The implication of this result on protein evolution is discussed.     

Time-resolved pH jump study of photochemical cleavage and release of carboxylic acids from α-keto amides

Time-resolved pH jump experiments, using laser flash photolysis and bromocresol green as an indicator, showed that photochemical cleavage and release of carboxylic acids from various α-keto amides derivatives in aqueous media occurs on the microsecond timescale (18-136 μs), depending on carboxylate leaving group ability.     

Synthesis of a dual-labeled probe of dimethyl lithospermate B with photochemical and fluorescent properties

Dimethyl lithosermate B (DLB) is a highly potent natural antioxidant and antidiabetic polyphenol with unknown mode of action. To determine its cellular targets, a photochemical and fluorescent dimethyl lithopermate B probe was designed and efficiently synthesized. The dual-labeled chemical probe for biological application was evaluated by UV and fluorescence to determine its electrochemical absorption and emission properties. This probe could be valuable for investigating ligand-protein interactions and subcellular localization.     

Self-organization in protein folding and the hydrophobic interaction

Self-organization is a critical aspect of living systems. During the folding of protein molecules, the hydrophobic interaction plays an important role in the collapse of the peptide chain to a compact shape. As the hydrophobic core tightens and excludes water, not only does the number of hydrophobic side chain contacts increase, but stabilization is further enhanced by an increase in strength of each hydrophobic interaction between side chains in the core. Thus, the self-organization of the protein folding process augments itself by enhancing the stability of the core against large-scale motions that would unfold the protein. Through calculations and computer simulations on a model four-helix bundle protein, we show how the strengthening of the hydrophobic interaction is crucial for stabilizing the core long enough for completion of the folding process and quantitatively manifests self-organizing dynamical behavior.     

Enzymes catalyzing protein folding and their cellular functions

In live cells, protein folding often cannot occur spontaneously, but requires the participation of helper proteins - molecular chaperones and foldases. The mechanisms employed by chaperones markedly increase the effectiveness of protein folding, but have no bearing on the rate of this process, whereas foldases actually accelerate protein folding by exerting a direct influence on the rate-limiting steps of the overall reaction. Two types of foldases are known, using different principles of action. Peptidyl-prolyl cis/trans isomerase and protein-disulfide isomerase catalyze the folding of every protein that needs isomerization of prolyl peptide bonds or formation and isomerization of disulfide bonds for proper folding. By contrast, some foldases operating in the periplasm of bacterial cells are specifically designed to help in the folding of substrate proteins whose primary structure does not contain sufficient information for correct folding. In this review, we discuss recent data on the catalytic mechanisms of both types of foldases, focusing specifically on how a catalyst provides the structural information required for the folding of a target protein. Comparative analysis of the mechanisms employed by two different periplasmic foldases is used to substantiate the notion that combinations of a protein which is unable to fold independently and a specific catalyst delivering the necessary steric information are probably designed to achieve some particular biological purposes. The review also covers the problem of participation of peptidyl-prolyl cis/trans isomerase in different cellular functions, highlighting the role of this enzyme in conformational rearrangements of folded native proteins.     

Laser flash photochemical oxidation to locate heme binding and conformational changes in myoglobin

Fast photochemical oxidation of proteins (FPOP), a means of protein footprinting, uses OH radicals to oxidize the solvent-exposed residues of proteins on a short time scale (∼1 μs). A 248 nm pulsed laser beam dissociates H₂O₂ (15 mM) into hydroxyl radicals. The radicals react with exposed and reactive amino acid residues (e.g., C, M, W, Y, F, H, L, I) in competition with quenching with a glutamine scavenger present in solution. We report here the use of FPOP to confirm that the F-helix is conformationally constrained in the holo form of myoglobin, whereas it is conformationally free in the apo form. The interpretation finds support in the differences in oxidation of various residues in the apo versus holo forms. The differential reactivity of leucine 137 suggests that it is part of a hinge region on the H-helix, enabling the binding pocket to close in the apo form. This is our second study that offers support that FPOP is capable of elucidating the conformational dynamics of proteins by obtaining information that may not be accessible by other methods.     

Solvent effect on the folding dynamics and structure of E6-associated protein characterized from ab initio protein folding simulations

Solvent effect on protein conformation and folding mechanism of E6-associated protein (E6ap) peptide are investigated using a recently developed charge update scheme termed as adaptive hydrogen bond-specific charge (AHBC). On the basis of the close agreement between the calculated helix contents from AHBC simulations and experimental results, we observed based on the presented simulations that the two ends of the peptide may simultaneously take part in the formation of the helical structure at the early stage of folding and finally merge to form a helix with lowest backbone RMSD of about 0.9 A? in 40% 2,2,2-trifluoroethanol solution. However, in pure water, the folding may start at the center of the peptide sequence instead of at the two opposite ends. The analysis of the free energy landscape indicates that the solvent may determine the folding clusters of E6ap, which subsequently leads to the different final folded structure. The current study demonstrates new insight to the role of solvent in the determination of protein structure and folding dynamics.