Compact Hydrophobic Core Research Articles

Characterization of collapsed states in the early stages of the refolding of hen lysozyme.

Early conformational states in the refolding of hen lysozyme from guanidine hydrochloride have been characterized by measuring both the fluorescence and the solvent exchange properties of tryptophan side chains. The indole proton occupancies indicate that at pH 5.5, 25 degrees C, half the protection against pulse labeling occurs in the dead time (4 ms) of the experiment, with the remaining protection developing with a time constant of 55 ms. Comparison of these data with the protection kinetics of backbone amides and with the fluorescence data provides evidence for hydrophobic collapse involving incorporation of tryptophan residues in a solvent-excluded state in advance of stable secondary structure formation. Analysis of the pH dependence of the indole hydrogen exchange protection is consistent with two or more structurally distinct collapsed states, and indicates that the generation of a correctly folded compact hydrophobic core is a key precursor to the formation of persistent native-like structure during refolding.

Molecular characterisation of a thermoactive β-1,3-glucanase from Oerskovia xanthineolytica

Molecular characterisation of a lytic thermoactive β-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed. A molecular mass of 27 195.6 ± 1.3 Da and an isoelectric point of 4.85 were determined by electrospray mass spectrometry and from its titration curve, respectively. Its thermoactivity profile shows it to be a heat-stable enzyme with a temperature optimum of 65°C. The secondary structure content of the protein was estimated by circular dichroism to be approx. 25% α-helix, 7% random coil, and 68% β-sheet and β-turn structure. Nuclear magnetic resonance spectra confirm the high content of β-structure. Furthermore, the presence of a compact hydrophobic core is indicated by the presence of slowly exchanging amide hydrogens and the enzyme's relatively high resistance to proteolysis. The N-terminal sequences of the intact protein and of a tryptic peptide each exhibit significant similarity to family 16 of glycosyl hydrolases whose overall fold is known to contain almost exclusively β-sheets and surface loops. Moreover, the sequenced tryptic peptide appears to encompass residues of the Oerskovia xanthineolytica glucanase active site, since it contains a portion of the family 16 active-site motif E-[L/I/V]-D-[L/I/V]-E.

Fast Protein Folding in the Hydrophobic–Hydrophilic Model within Three-Eighths of Optimal

We present performance-guaranteed approximation algorithms for the protein folding problem in the hydrophobic-hydrophilic model (Dill, 1985). Our algorithms are the first approximation algorithms in the literature with guaranteed performance for this model (Dill, 1994). The hydrophobic-hydrophilic model abstracts the dominant force of protein folding: the hydrophobic interaction. The protein is modeled as a chain of amino acids of length n that are of two types; H (hydrophobic, i.e., nonpolar) and P (hydrophilic, i.e., polar). Although this model is a simplification of more complex protein folding models, the protein folding structure prediction problem is notoriously difficult for this model. Our algorithms have linear (3n) or quadratic time and achieve a three-dimensional protein conformation that has a guaranteed free energy no worse than three-eighths of optimal. This result answers the open problem of Ngo et al. (1994) about the possible existence of an efficient approximation algorithm with guaranteed performance for protein structure prediction in any well-studied model of protein folding. By achieving speed and near-optimality simultaneously, our algorithms rigorously capture salient features of the recently proposed framework of protein folding by Sali et al. (1994). Equally important, the final conformations of our algorithms have significant secondary structure (antiparallel sheets, beta-sheets, compact hydrophobic core). Furthermore, hypothetical folding pathways can be described for our algorithms that fit within the framework of diffusion-collision protein folding proposed by Karplus and Weaver (1979). Computational limitations of algorithms that compute the optimal conformation have restricted their applicability to short sequences (length < or = 90). Because our algorithms trade computational accuracy for speed, they can construct near-optimal conformations in linear time for sequences of any size.

Polyelectrolyte Poly(tert-butyl acrylate)-block-poly(2-vinylpyridine) Micelles in Aqueous Media

Poly(tert-butyl acrylate)-block-poly(2-vinylpyridine), PBA-b-PVP, was prepared by living anionic polymerization. Polymolecular spherical micelles with compact hydrophobic PBA cores and protonated PVP shells in acid water were prepared by a dialysis from organic−aqueous mixtures and are stable if the pH is less than pH 4.8. The stretching of the PVP blocks in shells depends on pH and ionic strength of the solution and decreases significantly in a narrow pH region below 4.8. Small hydrophobic molecules, such as benzene, may be solubilized into micellar core swelling the core and releasing the segmental mobility of core-forming blocks. Two fluorimetric techniques for determination of pyrene partitioning between micelles and the aqueous phase were used. The first method is based on measurements of the ratio of intensities of the first and third vibrational bands, I1/I3, in the emission spectra of pyrene solubilized in a micellar solution as a function of micellar concentration. The second method is based on t...

Ion correlations in a micellar solution studied by small-angle neutron and x-ray scattering

We propose a general and consistent scheme for the analysis of small-angle x-ray and neutron data from quasispherical ionic surfactant micelles in aqueous solution. A two-shell model for the micellar particle structure factor is used, which models the structure in terms of a compact hydrophobic core and a hydrophilic outer shell. The hydrophilic shell contains head groups of the surfactant, water molecules of hydration, and a number of counterions, the number being determined by the counterion condensation mechanism. The partial structure factors for micelle-micelle, micelle-counterion, and counterion-counterion correlations are calculated by a multicomponent mean spherical model (MMSA) of Ronis and Khan. This model can be solved analytically under the conditions of penetrable spheres for pointlike counterions. The former condition is essential for treating the highly charged macroion system such as micelles by MMSA. This scheme is applied to a micellar solution composed of an ionic surfactant, cesium dodecyl sulfate. The presence of heavy counterions provides the sensitivity necessary to test the accuracy of the calculated degree of counterion condensation into the outer shell of the micelle.

Structure of the C3HC4 Domain by 1H-nuclear Magnetic Resonance Spectroscopy.: A New Structural Class of Zinc-finger

A recently identified sequence motif, referred to as "C3HC4" (also "RING finger" and "A Box") for its distinctive pattern of putative metal-binding residues, has been found in a wide range of proteins. In a previous paper we described the expression and purification of fragments encompassing this motif from the Vmw110 (IPC0) protein family. We showed that the equine herpes virus protein binds zinc ions and adopts a ββαβ fold. We now report the tertiary structure of this domain in solution, as determined by two-dimensional 1 H-NMR An amphipathic α-helix lies along one surface of a triple-stranded β-sheet. Four pairs of metal-binding residues sequester two zincs at distinct tetrahedral sites. The first and third pairs bind one metal ion, while the second and fourth pairs bind the other, forming an interleaved whole. The first and the fourth pairs are contained within two prominent, well-defined loops related by an approximate dyad symmetry. Conserved residues within the helix, sheet and loops contribute to a compact hydrophobic core. The region comprising the first two β-strands and the α-helix has remarkable structural similarity with a TFIIIA type of zinc finger, even though the C3HC4 domain appears not to bind specifically to DNA or RNA. Using site-directed mutagenesis we demonstrate that exposed polar side-chains of the C3HC4 α-helix are essential for trans-activation of gene expression by an intact herpes virus regulatory protein.

Crystal structure of recombinant human T-cell cyclophilin A at 2.5 A resolution.

The structure of the unligated human T-cell recombinant cyclophilin has been determined at 3 A resolution by multipole isomorphous replacement methods and refined at 2.5 A resolution to an R factor of 0.209. The root-mean-square errors of the bond lengths and bond angles are 0.013 A and 2.8 degrees from ideal geometry, respectively. The overall structure is a beta-barrel, consisting of eight antiparallel beta-strands wrapping around the barrel surface and two alpha-helices sitting on the top and the bottom closing the barrel. Inside the barrel, seven aromatic and other hydrophobic residues form a compact hydrophobic core. A loop of Lys-118 to His-126 and four beta-strands (B3-B6) constitute a pocket we speculate to be the binding site of cyclosporin A.

Structure of ricin B‐chain at 2.5 Å resolution

The heterodimeric plant toxin ricin has been refined to 2.5 A resolution. The B-chain lectin (RTB) is described in detail. The protein has two major domains, each of which has a galactose binding site. RTB has no regular secondary structure but displays several omega loops. Each RTB domain is made of three copies of a primitive 40 residue folding unit, which pack around a pseudo threefold axis. In each domain, galactose binds in a shallow cleft formed by a three residue peptide kink on the bottom and an aromatic ring on the top. At the back of the cleft, an aspartate forms hydrogen bonds to the C3 and C4 hydroxyls of galactose, whereas a glutamine bonds to the C4 alcohol, helping to define specific epimer binding. In addition to analyzing the sugar binding mechanism, the assembly of subdomain units around the pseudo threefold axis of each domain is described. The subdomains contribute conserved Trp, Leu, and Ile residues to a compact central hydrophobic core. This tight threefold binding probably drives the peptide folding and stabilizes the protein structure.

Chain statistics in micelles and bilayers: Effects of surface roughness and internal energy

A recently developed mean field (‘‘single-chain’’) theory for amphiphile chain organization and thermodynamics in micellar aggregates is applied to rotational isomeric state, model chains. The theory provides explicit, simple expressions for the probability distribution of chain conformations and related molecular and thermodynamic properties applicable to aggregates of arbitrary geometries. Bond order parameter profiles calculated from the theory for a planar bilayer, assuming a compact hydrophobic core, show very good agreement with experimental data and molecular dynamics simulations. For small spherical micelles comparison between theory and experiment suggest the existence of a somewhat rough (few angstroms wide) hydrocarbon–water interfacial region. Cylindrical aggregates reveal intermediate behavior. The extent of ‘‘surface roughness’’ is introduced into the theory via a density profile of the hydrophobic core which decreases gradually from the bulk liquid (compact core) density to zero. A series of calculations is presented to analyze the effects of internal chain (gauche/trans) energy and micellar geometry on the conformational and thermodyamic properties of the hydrocarbon chains. It is found that the internal energy plays only a secondary role, compared to the primary role of the packing constraints. (This is qualitatively consistent with our previous findings for approximate, ‘‘cubic,’’ model chains.) The conformational free energy cost associated with chain packing in aggregates is shown to depend on the micellar geometry (i.e., on the curvature of, and the average area per head group at, the hydrocarbon–water interface) and to be comparable with the surface (head group) contributions treated exclusively in the prevailing theories of surfactant self-assembly. Finally, a ‘‘corresponding-states’’ behavior is demonstrated for packed chains (in planar bilayers) by referencing all thermodynamic functions and configurational properties to those of the associated ‘‘free’’ chain.

On the molecular conformation of human haemopexin: II. Analysis of circular dichroic spectra

The circular dichroic (CD) spectra of haemopexin in the far ultraviolet region exhibit an atypical positive maximum at 231 nm, which prevents determination of the secondary structure by the usual methods. We have developed a modified analytical method by which it was possible to calculate the secondary structure of the protein (8% of α-helix, 10% of β-conformation) and the actual wavelength (229 nm) and intensity of the above-mentioned positive band. Measurements and analysis of the CD spectra of haemopexin in an alkaline medium gave information ascribing a major part of intensity of the band at 229 nm to the B Iu transition of Tyr; we believe that some other electronic transitions contribute to this band, too. An assignation of the individual extremes of the CD spectrum in the near ultraviolet region to the individual electronic transitions is propounded. The dichroic bands of Trp in this region and the temperature dependence of the CD spectra in the far ultraviolet region corroborate our idea that the haemopexin molecule contains a very compact hydrophobic peptide core. Similarities of the CD spectra of haemopexin to those of the haem. haemopexin complex throughout the ultraviolet region suggest a conformational likeness of these two molecules.

The amino acid sequence of the major parvalbumin of the whiting ( Gadus Merlangus)

1. 1. The amino acid sequence of the major parvalbumin of the Whiting has been determined; the polypeptide chain is made of 108 residues, the terminal amino acid group is acetylated, there is no disulfide bridges, the structure of the two calcium binding sites is preserved and the distribution along the polypeptide chain of the hydrophobic residues implicated in the compact hydrophobic core of the protein is also maintained. 2. 2. The comparison of this amino acid sequence with other parvalbumins indicates that it belongs to the β type and that within the Gadidae family two types of parvalbumins also occur.