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The folding of proteins in living cells may start during their synthesis when the poiypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from [alpha]-spectrin at sequential stages of elongation via in vivo ribosome-arrested [sup.15]N,[sup.13]C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like [beta]-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding. nascent chains | protein folding | ribosome doi/ 10.1073/pnas.0914300107

In an RNA interference screen interrogating regulators of mouse embryonic stem (ES) cell chromatin structure, we previously identified 62 genes required for ES cell viability. Among these 62 genes were Smc2 and -4, which are core components of the two mammalian condensin complexes. In this study, we show that for Smc2 and -4, as well as an additional 49 of the 62 genes, knockdown (KD) in somatic cells had minimal effects on proliferation or viability. Upon KD, Smc2 and -4 exhibited two pheno types that were unique to ES cells and unique among the ES cell--lethal targets: metaphase arrest and greatly enlarged interphase nuclei. Nuclear enlargement in condensin KD ES cells was caused by a defect in chromatin compaction rather than changes in DNA content. The altered compaction coincided with alterations in the abundance of several epigenetic modifications. These data reveal a unique role for condensin complexes in interphase chromatin compaction in ES cells. doi/ 10.1083/jcb.200908026

This study deals with the separation by capillary zone electrophoresis (CE) of hardly soluble synthetic polypeptides such as poly(L-alanine). The characterization of the molar mass distribution of these polypeptides with neutral lateral chain is a prerequisite for a good understanding of the relationship between their structure and their biological activity. Nevertheless, these polypeptides are hardly soluble in a limited number of solvents such as strong acids (trifluoroacetic acid, methanesulfonic acid) and hexafluoroisopropanol (HFIP). Highly resolutive separations of end-charged hydrophobic alanine oligomers (up to 110 residues) were obtained by CE in a mixed hydroorganic HFIP/water solvent system under optimal condilions. The analyzed polyalanines possess a single positive charge on the N-terminus of their chain, and the potential negative charge at the C-terminus of their chain is suppressed by replacement of C-terminal alanine for alaninol. A careful optimization of the different constituents of the electrolyte (HFIP content, nature and concentration of the acid, nature and concentration of the cellulose derivative) was required to limit solute adsorption onto the capillary wall and to achieve the characterization of the entire molar mass distribution. 10.1021/ac902211f

A system is described for the synthesis of polypeptides with novel folds in which existing sequence and structural data from bacterial proteins are used to assemble a pool of secondary structural elements. The results have shown that the protein evolution strategy has generated novel polypeptide sequences containing secondary structure.

A study is presented on the polypeptide backbone dynamics of hemoglobin (Hb) A in both deoxy and CO forms based on the Model-free analysis. It is found that some of them are not predicted by crystal structures of Hb A in deoxy and CO forms.

A natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] is examined to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. The results have shown that TRiC has an active role in rearranging the bound actin molecule.

A well-defined helical dimer native state of a two-stranded coiled-coil polypeptide is obtained by applying an off-lattice model. The effect of surface-immobilization of the polypeptide is analyzed for different surfaces and denaturation conditions.

The microscopic structure and photosensitive function of encapsulated dye in the single-wall carbon nanotubes (SWCNTs) are studied. The findings suggest that the photoexcitation of such dyes leads to the enhancement of transient absorption saturation in the nanotubes, which also proves the transfer of ultrafast energy from the dye to SWCNTs.

The analytic derivative of the electrostatic interaction between far separated fragments is developed and a number of restricted Hartree-Fock (RHF) geometry optimizations are performed by using the fragment molecular orbital method (FMO) and ab initio methods. The [alpha]-helix, [beta]-turn and extended conformers of a 10-residue polyanaline are analyzed and the good FMO accuracy is established.

Two models of the origin of novel polypeptide domains are proposed, based on 'exonization' of intron sequences and insertion-based polymorphisms between orthologous genes. These processes are discussed, also analyzing how each might participate in the evolutionary emergence of novel polypeptide domains.

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPlase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30[degrees]C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon. cold sensitivity | genetic suppression | nascent polypeptides | protein aggregation | protein export

Intrachain loop formation allows unfolded polypeptide chains to search for favorable interactions during protein folding. We applied triplet-triplet energy transfer between a xanthone moiety and naphthylalanine to directly measure loop formation in various unfolded polypeptide chains with loop regions consisting of polyserine, poly(glycine--serine) or polyproline. By combination of femtosecond and nanosecond laserflash experiments loop formation could be studied over many orders of magnitude in time from picoseconds to microseconds. The results reveal processes on different time scales indicating motions on different hierarchical levels of the free energy surface. A minor ( conformational substates | femtoseconds spectroscopy | peptide dynamics | protein folding | triplet-triplet energy transfer

Although protein structures are primarily encoded by their sequences, they are also critically dependent on environmental factors such as solvents and interactions with other molecules. Here we investigate how the folding-energy landscape of a short peptide is altered by interactions with another peptide, by performing atomistic replica-exchange molecular dynamics simulations of polyalanines in various environments. We analyzed the free-energy landscapes of [Ala.sup.7] and [Ala.sup.8] in isolation, near an [alpha]-helix template, and near a [beta]-strand template. The isolated [Ala.sup.7] and [Ala.sup.8] at 270 K were mainly in polyproline II helix conformations and in equilibrium between the [alpha]-helix and polyproline II helix, respectively, in harmony with the experiment. Interestingly, we found remarkably strong secondary-structure 'templating'; namely, the [alpha]-helix template enhanced [alpha]-helix conformation and the [beta]-strand template induced [beta]-strand conformation in the simulated [Ala.sup.8]. The [alpha]-helix template lowered the nearby dielectric constant, which strengthened hydrogen bonds in the simulated [Ala.sup.8], leading to [alpha]-helix stabilization. The [beta]-strand template provided hydrogen bond positions to the simulated [Ala.sup.8], sharply inducing [beta]-strand structure. With or without templates, the energy landscape of [Ala.sup.8] is always funnel-like and centered at the [alpha]-helix conformation, whereas entropic contribution disfavors the [alpha]-helix, leading to subtle competition. Secondary-structure templating may play a critical role in protein conformation dynamics in the cellular environment. energy landscape | generalized Born | polyproline II | protein folding | replica exchange

A simple two-state model for the dependence of the rate of folding of a polypeptide confined within a spherical cavity upon the size of the cavity relative to that of the polypeptide is described. The model generally predicts that decreasing the size of the cavity would increase the rate of refolding until the cavity becomes only slightly larger than the native state of the protein, at which point a further decrease in cavity size decreases the rate of refolding.

The use of [sup.1]H-[sup.1]H nuclear Overhauser effects (NOE) for structural studies of uniformly deuterated polypeptide chains in large structures is investigated by model calculations and NMR experiments. Detailed analysis of the evolution of the magnetization during [sup.1]H-[sup.1]H NOE experiments under slow-motion conditions shows that the maximal [sup.1]H-[sup.1]H NOE transfer is independent of the overall rotational correlation time, even in the presence of chemical exchange with the bulk water, provided that the mixing time is adjusted for the size of the structure studied. [sup.1]H-[sup.1]H NOE buildup measurements were performed for the 472-kDa complex of the 72-kDa cochaperonin GroES with a 400-kDa single-ring variant of the chaperonin GroEL (SR1). These experiments demonstrate that multidimensional NOESY experiments with cross-correlated relaxation-enhanced polarization transfer and transverse relaxation-optimized spectroscopy elements can be applied to structures of molecular masses up to several hundred kilodaltabs, which opens new possibilities for studying functional interactions in large maromolecular assemblies in solution. [sup.1]H-[sup.1]H NOE | GroE chaperonine system | NMR assignments | protein structure

Infrared multiphoton dissociation (IRMPD) of N-terminal sulfonated peptides improves de novo sequencing capabilities in a quadrupole ion trap mass spectrometer. Not only does IRMPD promote highly efficient dissociation of the N-terminal sulfonated peptides but also the entire series of y ions down to the [y.sub.1] fragment may be detected due to alleviation of the low-mass cutoff problem associated with conventional collisional activated dissociation (CAD) methods in a quadrupole ion trap. Commercial de novo sequencing software was applied for the interpretation of CAD and IRMPD MS/MS spectra collected for seven unmodified peptides and the corresponding N-terminal sulfonated species. In most cases, the additional information obtained by N-terminal sulfonation in combination with IRMPD provided significant improvements in sequence identification. The software sequence tag results were combined with a commercial database searching algorithm to interpret sequence information of a tryptic digest on [alpha]-casein s1. Energy-variable CAD studies confirmed a 30-40% reduction in the critical energies of the N-terminal sulfonated peptides relative to unmodified peptides. This reduction in dissociation energy facilitates IRMPD in a quadrupole ion trap.

The ion/molecule reactivity that reflects the radical character of product ions formed via gas-phase electron transfer to multiply protonated polypeptides is reported. The observations from the results indicate that ion/radical reactions could be used to probe the locations of radical sites in the undissociated electron transfer products as well as distinguish between c- and z-type ions.

The NMR analysis is used to explain the highly ordered, back-folded structure for human polypeptide YY (PYY) and human PYY3-36, which was found to be exactly similar to the classical pancreatic polypeptide (PP)-fold solution structure. The analysis proves that the PP-fold is extremely essential for establishing interactions with two subsites in the receptor for binding the N- and C-terminal ends of PYY, as both the segments are characterized by larger dynamics.

Supercritical fluid chromatography (SFC) provides a number of advantages over traditional HPLC such as speed, practical use of longer columns, a normal-phase retention mechanism, and reduced use of organic solvents. Yet, it has been a technique traditionally limited to relatively nonpolar compounds. The nature of SFC mobile and stationary phases did not allow the elution of ionic compounds or of peptides, except, in the latter case, for the most hydrophobic peptides. The characterization of peptides is critically important for drug discovery and development in the pharmaceutical industry, as well as for a variety of other important applications. Here, for the first time to our knowledge, we show that relatively large peptides (at least 40 mers), containing a variety of acidic and basic residues, can be eluted in SFC. We used trifluoroacetic acid as additive in a C[O.sub.2]/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. A 2-ethylpyridine bonded silica column, which was specifically developed for SFC, was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric detection.

The size and origin of the protein fold universe is of fundamental and practical importance. Analyzing randomly generated, compact sticky homopolypeptide conformations constructed in generic simplified and all-atom protein models, all have similar folds in the library of solved structures, the Protein Data Bank, and conversely, all compact, single-domain protein structures in the Protein Data Bank have structural analogues in the compact model set. Thus, both sets are highly likely complete, with the protein fold universe arising from compact conformations of hydrogen-bonded, secondary structures. Because side chains are represented by their [C.sup.[beta]] atoms, these results also suggest that the observed protein folds are insensitive to the details of side-chain packing. Sequence specificity enters both in fine-tuning the structure and thermodynamically stabilizing a given fold with respect to the set of alternatives. Scanning the models against a three-dimensional active-site library, close geometric matches are frequently found. Thus, the presence of active-site-like geometries also seems to be a consequence of the packing of compact, secondary structural elements. These results have significant implications for the evolution of protein structure and function. evolution | Protein Data Bank | protein folding | protein structure prediction

Peptide scaffolds are developed with the use of heterochiral Monte Carlo (HCMC) for interacting with a molecular target, a left-handed alpha-helix. The HCMC approach concurrently seeks to optimize a peptide sequence, its internal conformation, and its docked conformation with a target surface.

The anthrax toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to anthrax toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization.

The preparation and characterization of charged, amphiphilic block copolypeptides that form stable vesicles and micelles in aqueous solution are reported. The vesicular assemblies reveal dynamic properties, indicating a high degree of membrane fluidity.

The aqueous, lyotropic liquid-crystalline state pattern of the alpha-helical polypeptide is studied using optical microscopy and X-ray scattering. It was observed that the solutions formed cholesteric liquid crystals at volume fractions that reduced with increasing average chain length.

Several molecular dynamics simulations are conducted to explain the significance of polypeptide backbone conformation in the formation of different macromolecules in nisin mimics. The study has been shown to be extremely beneficial for the formation of macrocyclic peptides, as well as lantibiotic antigens.

NMR spectroscopy, structure calculation and energy minimization are applied to determine the three-dimensional structure of polytheonamide B (pTB), a highly cytotoxic polypeptide which is one of the most unusual nonribosomal peptides of sponge origin. The pTB molecules are found to form transmembrane channels that permeate monovalent cations similar to gramicidin A channels and the strong cytotoxicity of pTB could be attributed to its ability to form single molecule channels through biological membranes.

Sulfite oxidizing enzymes (SOEs) are studied by using kinetic measurements, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements and site-directed mutagenesis on active site residues of SOEs and of the flexible polypeptide tether that connects the heme and molybdenum domains of human sulfite oxidase (SO). Rapid kinetic studies of plant SO (PSO) are discussed.

Well-characterized elastin-like polypeptides (ELP) containing three hydrophobic domains flanking two cross-linking domains is used to describe the effects of temperature, polypeptide concentration, and solution conditions on the kinetics of self-assembly of an elastin-like polypeptide tropoelastin. The results provide insight into the possible mechanisms for the spontaneous organization of such ELPs into extended networks.

A study was conducted to demonstrate the role of zinc found at millimolar concentrations in the secretory granule towards inhibition of human islet amyloid polypeptide (hIAPP) amyloid fibrillogenesis at concentrations similar to those found in the extracellular environment. The inhibition of the formation of aggregated and toxic forms of hIAPP by zinc provides insight into the possible mechanism for the linkage between deleterious mutations in the SLC30A8 zinc transporter, which transports zinc into the secretory granule, and type II diabetes.

A study was conducted to understand the disruption of the covalent bond between the cysteine and flavin of Phot1LOV2-linker. The unfolded conformation of the linker folds with a time constant of 13 ms was found to be considerably slower than the helix formation rate measured for an [alpha]-helical polypeptide in solution.

The semisynthesis of a bacterial nonselective cation channel, NaK polypeptide that is assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction is reported. Functional characterization of the folded semisynthetic NaK channels indicates that it is functionally similar to the wild-type protein and the presence of a negatively charged residue in the vicinity of the ion binding sites is necessary for optimal flux of ions through the NaK channel.

The crystal structure of the human nascent polypeptide-associated complex (NAC) domain was characterized to demonstrate the kinetics of human NAC dimerization. The results identified a region in the NAC domain of the human NAC [alpha]-subunit as a new nucleic acid-binding region which is blocked from binding nucleic acids in the heterodimeric complex by a helix region in the [beta]-subunit.

The synthesis and self-assembly of the new class of peptide chimera with a series of amphiphilic polypeptide-b-designed peptides are described in which the hydrophobic block is poly([gamma]-benzyl L-glutamate) (PBLG) and the hydrophilic block is a coiled-coil forming peptide (E). The studies have shown that as the structures have encapsulated water-soluble compounds and the surfaces are functionalized by the coiled-coil binding, these peptide-based nanocapsules have acted as delivery vehicles to specific targets in the body.

A proof-of-principle study which could be used to demonstrate the efficient folding, with concomitant formation of the correct disulfides, of an isolated polypeptide insulin precursor of defined covalent structure, is reported. The defined chemical tethering principle could form the basis of a practical, high yield total synthesis of insulin and analogues.

Leucyl-trRNA synthetase (LeuRS) is identified as a target for a novel class of boron-containing small molecules that bind to its editing active site. The human cytoplasmic enzyme (hscLeuRS) editing active site is present in a discrete domain called the connective polypeptide 1 domain (CP1), where mischarged tRNA binds for hydrolysis of the noncognate amino acid.