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At least 397 records · Page 22Linked to original sources

PubMed2005

Chemical synthesis of the RGD-protein decorsin: Pro-->Ala replacement reduces protein thermostability.

Decorsin is a 39-residue polypeptide chain, crosslinked by three disulfide bridges, that strongly inhibits platelet aggregation. We report the chemical synthesis and characterization of analogs of decorsin with the aim of investigating the role of proline residues in protein structure, stability and biological activity. Decorsin analogs have been synthesized in which one (P23A and P24A decorsin) or two (P23,24A decorsin) proline residues have been substituted by alanine. The crude synthetic polypeptides were purified by reversed-phase HPLC in their reduced form and allowed to refold oxidatively to their disulfide-crosslinked species. The homogeneity of the synthetic mini-proteins, and also the correct pairing of the three disulfide bridges, were established by a number of analytical criteria, including fingerprinting analysis of the refolded synthetic analogs by using thermolysin and proteinase K as proteolytic enzymes. Replacement of proline by alanine results in a significant and cumulative decrease of the high thermal stability (Tm 74 degrees C) of native decorsin. The mono-substituted analogs display a Tm of 66-67 degrees C, while the double-substituted analog a Tm of 50 degrees C. On the other hand, the overall secondary and tertiary structures were not affected by the Pro-->Ala exchanges, as judged from circular dichroism measurements. Platelet aggregation assays established that the proline substitutions do not impair significantly the biological activity of decorsin. The results of this study clearly indicate that proline residues contribute significantly to the protein thermal stability. Our results are in line with the 'proline rule', previously advanced for explaining the unusual thermal stability of thermophilic enzymes, which usually show an enhanced content of proline residues with respect to their mesophilic counterparts.

Alanine↗

PubMed2003

A combined atomic force microscopy and molecular dynamics simulation study on a plastocyanin mutant chemisorbed on a gold surface.

A mutant of copper plastocyanin, covalently bound to an Au (111) surface through an engineered disulfide bridge, was investigated in aqueous medium by atomic force microscopy (AFM) and molecular dynamics (MD) simulations. Tapping-mode AFM images revealed adsorption of single molecules which are homogeneously distributed over the substrate and strongly bound to gold and display uniform lateral size. A statistical analysis of the height of the macromolecules on the gold substrate evidenced a distribution around a mean value consistent with that expected from the crystallographic data and with a relatively large standard deviation. A 10-ns classical MD simulation of mutated plastocyanin, hydrated by a layer of water, covalently bound to a gold surface by one or two sulfur atoms, was performed. The simulations indicate that the bound protein retains, in both cases, its overall tertiary structure during the dynamic evolution. Moreover, the macro-molecule can assume different orientations with respect to the gold substrate, which give rise to a distribution of heights on the gold substrate. Experimental and MD simulation results are compared and discussed in connection with the topological and dynamical properties of the protein system.

Adsorption↗

PubMed2005

Prediction of "hot spots" of aggregation in disease-linked polypeptides.

BACKGROUND: The polypeptides involved in amyloidogenesis may be globular proteins with a defined 3D-structure or natively unfolded proteins. The first class includes polypeptides such as beta2-microglobulin, lysozyme, transthyretin or the prion protein, whereas beta-amyloid peptide, amylin or alpha-synuclein all belong to the second class. Recent studies suggest that specific regions in the proteins act as "hot spots" driving aggregation. This should be especially relevant for natively unfolded proteins or unfolded states of globular proteins as they lack significant secondary and tertiary structure and specific intra-chain interactions that can mask these aggregation-prone regions. Prediction of such sequence stretches is important since they are potential therapeutic targets. RESULTS: In this study we exploited the experimental data obtained in an in vivo system using beta-amyloid peptide as a model to derive the individual aggregation propensities of natural amino acids. These data are used to generate aggregation profiles for different disease-related polypeptides. The approach detects the presence of "hot spots" which have been already validated experimentally in the literature and provides insights into the effect of disease-linked mutations in these polypeptides. CONCLUSION: The proposed method might become a useful tool for the future development of sequence-targeted anti-aggregation pharmaceuticals.

Amyloid beta-Peptides↗

PubMed1997

The L7/L12 ribosomal domain of the ribosome: structural and functional studies.

The L7/L12 protein forms a functionally important domain in the ribosome. This domain is involved in interaction with translation factors during protein biosynthesis. The tertiary and quaternary structure of the L7/L12 protein was established as a result of intensive studies in solution and in the ribosome. The conformational changes of L7/L12, the elongation factors Tu and G and other ribosomal proteins were traced by different experimental techniques. These changes occur upon interaction of the ribosome with the elongation factors and depend on GTP hydrolysis in accordance with the functional states of the ribosome.

Bacterial Proteins↗

PubMed2003

Low-frequency modes of peptides and globular proteins in solution observed by ultrafast OHD-RIKES spectroscopy.

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.

Alanine↗

PubMed1999

Helix capping in the GCN4 leucine zipper.

Capping interactions associated with specific sequences at or near the ends of alpha-helices are important determinants of the stability of protein secondary and tertiary structure. We investigate here the role of the helix-capping motif Ser-X-X-Glu, a sequence that occurs frequently at the N termini of alpha helices in proteins, on the conformation and stability of the GCN4 leucine zipper. The 1.8 A resolution crystal structure of the capped molecule reveals distinct conformations, packing geometries and hydrogen-bonding networks at the amino terminus of the two helices in the leucine zipper dimer. The free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is -1.2 kcal/mol, evaluated from thermal unfolding experiments. A single cap thus contributes appreciably to stabilizing the terminated helix and thereby the native state. These results suggest that helix capping plays a further role in protein folding, providing a sensitive connector linking alpha-helix formation to the developing tertiary structure of a protein.

Amino Acid Sequence↗

PubMed2003

Direct observation of photolysis-induced tertiary structural changes in hemoglobin.

Human Hb, an alpha2beta2 tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the alpha subunit than in the beta subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state beta heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.

Carboxyhemoglobin↗

PubMed1995

Conserved nonplanar heme distortions in cytochromes c.

A nonplanar distortion of the heme of c-type cytochromes is conserved in the proteins isolated from diverse species based upon a comprehensive analysis of available high-resolution X-ray crystal structures. This distortion is induced through the cysteine thioether linkages between the porphyrin pyrrole groups and the polypeptide and results in an asymmetric pyrrole distortion. This asymmetry in the heme distortion is also conserved. For other heme proteins which lack these covalent bonds, nearly planar porphyrins are observed. Resonance Raman evidence indicates that nonplanar distortion of porphyrins containing metals, like iron, with large core sizes (> or = 2.00 A) is energetically unfavorable and can occur only in the presence of significant environmental perturbations. Further, energy minimization and dynamics calculations on the ferric form of yeast iso-1-cytochrome c, starting from the crystallographic coordinates and using a molecular mechanics force field which accurately reproduces nonplanar distortions in metalloporphyrins, suggest that this distortion is indeed maintained by the protein tertiary structure. It is proposed that this protein-linked heme distortion modulates electron transfer function through modification of redox potentials of the porphyrin ring and the protein binding properties of c-type cytochromes.

Cytochrome c Group↗

PubMed2002

SnapDRAGON: a method to delineate protein structural domains from sequence data.

We describe a method to identify protein domain boundaries from sequence information alone based on the assumption that hydrophobic residues cluster together in space. SnapDRAGON is a suite of programs developed to predict domain boundaries based on the consistency observed in a set of alternative ab initio three-dimensional (3D) models generated for a given protein multiple sequence alignment. This is achieved by running a distance geometry-based folding technique in conjunction with a 3D-domain assignment algorithm. The overall accuracy of our method in predicting the number of domains for a non-redundant data set of 414 multiple alignments, representing 185 single and 231 multiple-domain proteins, is 72.4 %. Using domain linker regions observed in the tertiary structures associated with each query alignment as the standard of truth, inter-domain boundary positions are delineated with an accuracy of 63.9 % for proteins comprising continuous domains only, and 35.4 % for proteins with discontinuous domains. Overall, domain boundaries are delineated with an accuracy of 51.8 %. The prediction accuracy values are independent of the pair-wise sequence similarities within each of the alignments. These results demonstrate the capability of our method to delineate domains in protein sequences associated with a wide variety of structural domain organisation.

Algorithms↗

PubMed1997

Exploring the folding free energy surface of a three-helix bundle protein.

The multidimensional free energy surface for a small fast folding helical protein is explored based on first-principle calculations. The model represents the 46-residue segment from fragment B of staphylococcal protein A. The relationship between collapse and tertiary structure formation, and the order of collapse and secondary structure formation, are investigated. We find that the initial collapse process gives rise to a transition state with about 30% of the native tertiary structure and 50-70% of the native helix content. We also observe two distinct distributions of native helix in this collapsed state (Rg approximately 12 A), one with about 20% of the native helical hydrogen bonds, the other with near 70%. The former corresponds to a local minimum. The barrier from this metastable state to the native state is about 2 kBT. In the latter case, folding is essentially a downhill process involving topological assembly. In addition, the order of formation of secondary structure among the three helices is examined. We observe cooperative formation of the secondary structure in helix I and helix II. Secondary structure in helix III starts to form following the formation of certain secondary structure in both helix I and helix II. Comparisons of our results with those from theory and experiment are made.

Hydrogen Bonding↗

PubMed2002

Integral membrane protein biosynthesis: why topology is hard to predict.

Integral membrane protein biogenesis requires the coordination of several events: accurate targeting of the nascent chain to the membrane; recognition, orientation and integration of transmembrane (TM) domains; and proper formation of tertiary and quaternary structure. Initially unanticipated inter- and intra-protein interactions probably mediate each stage of biogenesis for single spanning, polytopic and C-terminally anchored membrane proteins. The importance of these regulated interactions is illustrated by analysis of topology prediction algorithm failures. Misassigned or misoriented TM domains occur because the primary sequence and overall hydrophobicity of a single TM domain are not the only determinants of membrane integration.

Algorithms↗

PubMed1995

Computer modeling of protein folding: conformational and energetic analysis of reduced and detailed protein models.

Recently we developed methods to generate low-resolution protein tertiary structures using a reduced model of the protein where secondary structure is specified and a simple potential based on a statistical analysis of the Protein Data Bank is employed. Here we present the results of an extensive analysis of a large number of detailed, all-atom structures generated from these reduced model structures. Following side-chain addition, minimization and simulated annealing simulations are carried out with a molecular mechanics potential including an approximate continuum solvent treatment. By combining reduced model simulations with molecular modeling calculations we generate energetically competitive, plausible misfolded structures which provide a more significant test of the potential function than current misfolded models based on superimposing the native sequence on the folded structures of completely different proteins. The various contributions to the total energy and their interdependence are analyzed in detail for many conformations of three proteins (myoglobin, the C-terminal fragment of the L7/L12 ribosomal protein, and the N-terminal domain of phage 434 repressor). Our analysis indicates that the all-atom potential performs reasonably well in distinguishing the native structure. It also reveals inadequacies in the reduced model potential, which suggests how this potential can be improved to yield greater accuracy. Preliminary results with an improved potential are presented.

Algorithms↗

PubMed2003

Hydrophobicity of transmembrane proteins: spatially profiling the distribution.

A hallmark of soluble globular protein tertiary structure is a hydrophobic core and a protein exterior populated predominantly by hydrophilic residues. Recent hydrophobic moment profiling of the spatial distribution of 30 globular proteins of diverse size and structure had revealed features of this distribution that were comparable. Analogous profiling of the hydrophobicity distribution of the alpha-helical buried bundles of several transmembrane proteins, as the lipid/protein interface is approached from within the bilayer, reveals spatial hydrophobicity profiles that contrast with those obtained for the soluble proteins. The calculations, which enable relative changes of hydrophobicity to be simply identified over the entire spatial extent of the multimer within the lipid bilayer, show the accumulated zero-order moments of the bundles to be mainly inverted with respect to that found for the soluble proteins. This indicates a statistical increase in the average residue hydrophobic content as the lipid bilayer is approached. This result differs from that of a relatively recent calculation and qualitatively agrees with earlier calculations involving lipid exposed and buried residues of the alpha-helices of transmembrane proteins. Spatial profiling, over the entire spatial extent of the multimer with scaled values of residue hydrophobicity, provides information that is not available from calculations using lipid exposure alone.

Animals↗

PubMed2003

Comparative structural studies of Vpu peptides in phospholipid monolayers by x-ray scattering.

Vpu is an 81-residue HIV-1 accessory protein, its transmembrane and cytoplasmic domains each responsible for one of its two functions. Langmuir monolayers of phospholipid incorporating a membrane protein with a unidirectional vectorial orientation, on a semiinfinite aqueous subphase, provide one "membranelike" environment for the protein. The cytoplasmic domain's interaction with the surface of the phospholipid monolayer in determining the tertiary structure of the peptide within the monolayer was investigated, employing a comparative structural study of Vpu with its submolecular fragments Tm and TmCy truncated to different extents in the cytoplasmic domain, via synchrotron x-ray scattering utilizing a new method of analysis. Localizations of the transmembrane and cytoplasmic domains within the monolayer profile structure were similar for all three proteins, the hydrophobic transmembrane helix within the hydrocarbon chain region tilted with respect to the monolayer plane and the helices of the cytoplasmic domains lying on the surface of the headgroups parallel to the monolayer plane. The thickness of the hydrocarbon chain region, determined by the tilt of the hydrocarbon chains and transmembrane domain with respect to the monolayer plane, was slightly different for Tm, TmCy, and Vpu systematically with protein/lipid mole ratio. Localization of the helices in the cytoplasmic domains of the three proteins relative to the headgroups depends on their extents and amphipathicities. Thus, the interaction of the cytoplasmic domain of Vpu on the surface may affect the tilt of the transmembrane helix within the hydrocarbon chain region in determining its tertiary structure in the membrane.

Amino Acid Sequence↗

PubMed1997

Granulocyte-colony stimulating factor maintains a thermally stable, compact, partially folded structure at pH2.

At acidic pH many proteins exist in a partially unfolded form, called the "A" state. This is defined as a flexible, expanded structure with well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induced denaturation. Granulocyte-colony stimulating factor (G-CSF), a four-helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.0. We therefore examined the conformation and thermal unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stoke's radius even at pH 2.0. The thermal-induced denaturation of G-CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some alpha-helical structure persists even at 86 degrees C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G-CSF at pH 2.0 is thus revealed as compact, with a unique, three-dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the "A" state.

Circular Dichroism↗

PubMed1998

Tertiary structure prediction of mixed alpha/beta proteins via energy minimization.

We describe an improved algorithm for protein structure prediction, assuming that the location of secondary structural elements is known, with particular focus on prediction for proteins containing beta-strands. Hydrogen bonding terms are incorporated into the potential function, supplementing our previously developed residue-residue potential which is based on a combination of database statistics and an excluded volume term. Two small mixed alpha/beta proteins, 1-CTF and BPTI, are studied. In order to obtain native-like structures, it is necessary to allow the beta-strands in BPTI to distort substantially from an ideal geometry, and an automated algorithm to carry this out efficiently is presented. Simulated annealing Monte Carlo methods, which contain a genetic algorithm component as well, are used to produce an ensemble of low-energy structures. For both proteins, a cluster of structures with low RMS deviation from the native structure is generated and the energetic ranking of this cluster is in the top 2 or 3 clusters obtained from simulations. These results are encouraging with regard to the possibility of constructing a robust procedure for tertiary folding which is applicable to beta-strand containing proteins.

Algorithms↗

PubMed2006

Control of macromolecular structure and function using covalently attached double-stranded DNA constraints.

The biophysical properties of DNA suggest its use for applications beyond serving as the genetic material. Several recent reports describe the use of covalently attached double-stranded DNA for controlling the structures of other macromolecules such as protein and RNA. These exploitations of DNA rigidity are conceptually distinct from many other studies in the area of "DNA nanotechnology". Double-stranded DNA constraints provide a means of introducing selective tension onto other molecules. This should facilitate fundamental investigations of macromolecular folding landscapes and tertiary interactions, as well as allow study of the mechanotransduction of biochemical signals. Use of a DNA constraint as the key element of a sensor has already been demonstrated, and such applications will be enhanced by improvements in the signal readout methods. If practical challenges such as delivery and stability can be addressed, these new efforts may also enable development of selective sensors for in vivo applications.

Base Sequence↗

PubMed2001

Kinetic and crystallographic analyses support a sequential-ordered bi bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase.

Glucose-1-phosphate thymidylyltransferase is the first enzyme in the biosynthesis of dTDP-l-rhamnose, the precursor of l-rhamnose, an essential component of surface antigens, such as the O-lipopolysaccharide, mediating virulence and adhesion to host tissues in many microorganisms. The enzyme catalyses the formation of dTDP-glucose, from dTTP and glucose 1-phosphate, as well as its pyrophosphorolysis. To shed more light on the catalytic properties of glucose-1-phosphate thymidylyltransferase from Escherichia coli, specifically distinguishing between ping pong and sequential ordered bi bi reaction mechanisms, the enzyme kinetic properties have been analysed in the presence of different substrates and inhibitors. Moreover, three different complexes of glucose-1-phosphate thymidylyltransferase (co-crystallized with dTDP, with dTMP and glucose-1-phosphate, with d-thymidine and glucose-1-phosphate) have been analysed by X-ray crystallography, in the 1.9-2.3 A resolution range (R-factors of 17.3-17.5 %). The homotetrameric enzyme shows strongly conserved substrate/inhibitor binding modes in a surface cavity next to the topological switch-point of a quasi-Rossmann fold. Inspection of the subunit tertiary structure reveals relationships to other enzymes involved in the biosynthesis of nucleotide-sugars, including distant proteins such as the molybdenum cofactor biosynthesis protein MobA. The precise location of the substrate relative to putative reactive residues in the catalytic center suggests that, in keeping with the results of the kinetic measurements, both catalysed reactions, i.e. dTDP-glucose biosynthesis and pyrophosphorolysis, follow a sequential ordered bi bi catalytic mechanism.

Amino Acid Sequence↗