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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.04.065 Byline: Srinivas Honnappa (1), Susana Montenegro Gouveia (2), Anke Weisbrich (1), Fred F. Damberger (3), Neel S. Bhavesh (3), Hatim Jawhari (1), Ilya Grigoriev (2), Frederik J.A. van Rijssel (2), Ruben M. Buey (1), Aleksandra Lawera (2), Ilian Jelesarov (4), Fritz K. Winkler (1), Kurt Wuthrich (3)(5), Anna Akhmanova (2), Michel O. Steinmetz (1) Keywords: CELLBIO Abstract: Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general 'microtubule tip localization signal' (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process. Author Affiliation: (1) Biomolecular Research, Structural Biology, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland (2) Department of Cell Biology, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands (3) Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland (4) Biochemisches Institut der Universitat Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland (5) Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA Article History: Received 10 November 2008; Revised 9 March 2009; Accepted 27 April 2009 Article Note: (miscellaneous) Published: July 23, 2009

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.

Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 meta-bolically labeled ([sup.14]N/[sup.15]N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.

A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [[gamma]([sup.18][O.sub.4])-ATP], has been developed. The ability to use [gamma]([sup.18][O.sub.4])-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of [gamma]([sup.18][O.sub.4])-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and [gamma]([sup.18][O.sub.4])-ATP. Standard peptides phosphorylated using [gamma]([sup.18][O.sub.4])-ATP via in vitro kinase reactions showed no exchange loss of [sup.18]O with [16.sup]O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins.

The molecular dynamics simulation studies of HNP defensins sequences that are human antimicrobial peptides produced in response to microbial invasion are reported. The results provide insight into their relationship, structural dissimilarities, and different microbial actions.

An effective and flexible computational approach is described and its usefulness and reliability is demonstrated by the tuning of the equilibrium between [3.sub.10] and [alpha]-helices of polypeptides by different solvents. The generality of this computational strategy and its applicability in effective and user-friendly computer codes have paved the way toward applications in the field of biomolecules and other complex systems.

In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. in some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universal-code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61-64) and YxCxxxF (amino acids 124-131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons. ciliated protozoa | dual gene reporter system | eukaryote class-1 polypeptide release factors | interdomain and intradomain protein chimeras | stop codon decoding

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 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.

The pathway of oxidative folding of bovine [alpha]-interferon (BoINF-[alpha]) a single Polypeptide is investigated. It is observed that the folding mechanism of BoINF-[alpha] appears compatible with the framework model.

Appropriate positioning and the judicious choice of nearby amino acid residues are demonstrated to lead to >900-fold differences in reactivity of otherwise identical functional groups in short peptides. The described ribosomes and nonribosomal peptide synthetases active site models do not approach the efficiencies of natural enzymes.

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 experimental and calculation data for short alanine-based peptides embedded in water is used to model the mechanism of helix folding and unfolding and to calculate microscopically the free energy factors of alanine in the frame of helix coil conformational integrals. Monte Carlo numerical simulations of thermodynamics and kinetics for the 21 amino acid [alpha]-helical polypeptide Ac-A5(AAARA)3A-NMe are conducted and found to be in an agreement with the experimental results.

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

An efficient strategy for immobilizing proteins on a gold surface was developed by employing the gold binding polypeptide (GBP) as a fusion partner. Using the enhanced green fluorescent protein (EGFP), severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVme), and core streptavidin (cSA) of Streptomyces avidinii as model proteins, specific immobilization of the GBP-fusion proteins onto the gold nanoparticles and generation of protein nanopatterns on the bare gold surface were demonstrated. The GBP-fused SCVme bound to gold nanoparticles successfully interacted with its antibody and showed changes in absorbance and color, allowing efficient diagnosis of SARS-CoV. The fusion proteins could be successfully immobilized on the gold surface by nanopatterning and microcontact printing as examined by atomic force microscopy and surface plasmon resonance analysis. The poly(dimethylsiloxane) microfluidic channels were created on the gold surface and were used for antigen-antibody and DNA--DNA interaction studies. Specific immobilization of GBP--EGFP fusion protein and its interaction with the antibody in the microchannels could be demonstrated. By immobilizing the DNA probe through the use of GBP-fused cSA, specific hybridization of the target DNA prepared from Salmonella could also be achieved. The GBP-fusion method allows immobilization of proteins onto the gold surface without surface modification and in bioactive forms suitable for studying protein--protein, DNA--DNA, and other biomolecular interaction studies. Furthermore, these studies can be carried out in a microfluidic system, which allows high-throughput analysis of biomolecular interactions.

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.

A series of engineered, variable-length de novo polypeptides is used to discreetly immobilize luminescent semiconductor nanocrystals or quantum dots (QDs) onto functional surfaces. The versatility of this immobilization strategy is highlighted by the creation of a variety of one- and two-dimensional polypeptide-QD structures as well as a self-assembled surface-immobilized fluorescence resonance energy transfer (FRET)-based utrient sensor.

A new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups is demonstrated, using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength.

Limited proteolysis is used to carry out an extensive study of the conformation of Toxoplasma gondii ferrodoxin-NADP(super +) reductase (FNR). It is found that two flexible polypeptide regions of Toxoplasma gondii FNR are involved in ferrodoxin (Fd) interaction but have opposite roles in modulating the binding affinity for the protein ligand.

Several novel, fully synthetic, carbohydrate-based antitumor vaccines are assembled where each construct consists of multiple cancer-related antigens displayed on a single polypeptide backbone. Advances in synthetic methodology have allowed for the incorporation of a complex oligosaccharide terminating in a sialic acid residue as one of the carbohydrate antigens.

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.

Water-soluble alpha-helical polypeptides were used in a study to prepare silica coated hexagonal single crystal platelets in concentrated solutions. The helices were also used to align samples giving monoliths composed of highly oriented layers of platelets, thus allowing preparation of composites with good structural order and complexity through a simple biomimetic process.

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

A study is presented to establish that the herpes simplex virus-1 (HSV-1) ICP6 polypeptide associates with eIF4G, the cap-binding protein, to promote eIF4F complex assembly. The factors which are capable of interacting with eIF4G and promoting eIF4G complex assembly may play important roles in a variety of processes where translation complexes need to be remodeled or assembled on populations of newly synthesized or derepressed mRNAs.

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.

Several experiments have suggested that newly synthesized polypeptide chains can adopt helical structures deep within the ribosome exit tunnel. We hypothesize that confinement in the roughly cylindrical tunnel can entropically stabilize [alpha]-helices. The hypothesis is validated by using theory and simulations of coarse-grained off-lattice models. The model helix, which is unstable in the bulk, is stabilized in a cylindrical cavity provided the diameter (D) of the cylinder exceeds a critical value D *. When D < D * both the helical content and the helix-coil transition temperature ([T.sub.f]) decrease abruptly. Surprisingly, we find that the stability of the [alpha]-helix depends on the number (N) of amino acid residues. Entropic stabilization, as measured by changes in [T.sub.f], increases nonlinearly as N increases. The simulation results are in quantitative agreement with a standard helix-coil theory that takes into account entropy cost of confining a polypeptide chain in a cylinder. The results of this work are in qualitative accord with most of the findings of a recent experiment in which N-dependent ribosome-induced helix stabilization of transmembrane sequences was measured by fluorescence resonance energy transfer. confinement effects | cylindrical pores | entropic stabilization folding

The isolated B-chain peptide of bovine insulin is shown to form fibrils at both acidic or neutral pH. The effects of cross-seeding were examined to investigate the interaction between the two polypeptides. Results of the investigation demonstrate the significant 'species' barrier in amyloid fibril formation between fibrillation induced by different fibrils.

Apolipoprotein (apo) A-I- mutants were constructed for FRET studies to distinguish between two possible lipid-free conformers. Results of the examination indicate the presence of a discrete globular bundle conformation for lipid-free apo A-I.

Turbidity and thioflavine-T fluorescence studies show that aggregation of islet amyloid polypeptide (IAPP) is faster at a lower pH (4.0 plusmn 0.3) than at high pH (8.8 plusmn 0.3). Results indicate differences in the morphology of the deposits formed at high and low pH.

The significant effects of the structure of both cross-linking and hydrophobic domains on the assembly of a series of a well-defined recombinant polypeptides based on native-elastin like human elastin sequences is examined. It is suggested that the major effect of trifluoroethanol (TFL) to promote organized self-assembly of the polypeptides is related to direct effects of the cosolvent on hydrophobic domains.

The study is conducted to know precisely the binding site that mediates FVIIIa interaction with activated platelets resides within residues 2303-2332 in the C2 domain of FVIII. The conclusion states that although the direct interaction of FVIIIa with the platelet surface appears to be mediated exclusively by the C2 domain (residues 2302-2332), protein interactive sites within the light chain ( A3-C1-C2) and the heavy chain (A1-A2) affect both the stoichiometry and the affinity of the FVIIIa binding.

Mutations G473D and A208D were identified in patients with isolated sulphide oxidase (SO) deficiency, and the equivalent amino acids are localized to the vicinity of the molybdopterin active site in the X-ray structure of chicken SO. The results clearly implicate the importance go G473 and A208 in controlling the rates of the IET process in human SO and allow the deleterious impact of these mutations to be rationalized.

Circular dichroism (CD) and fluorescence spectroscopy are used to investigate the molecular details of the interaction in islet amyloid polypeptide (IAPP) with lipid membranes of varying composition. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.

The energetics are compared using correlated ab inito calculations when the CH and NH groups of the model dipeptide CHONHCH2CONH2 are each allowed to form a H-bond with the proton acceptor O of a peptide group. The immersion of the depeptide-peptide pair in a model solvent wakens both the types of H-bonds and in a fairly manner.

Various reagent anions capable of converting polypeptide cations to anions via ion/ion reactions have been investigated. The major charge inversion reaction channels include multiple proton transfer and adduct formation. Dianions composed of sulfonate groups as the negative charge carriers show essentially exclusive adduct formation in converting protonated peptides and proteins to anions. Dianions composed of carboxylate groups, on the other hand, show far more charge inversion via multiple proton transfer, with the degree of adduct formation dependent upon both the size of the polypeptide and the spacings between carboxylate groups in the dianion. More highly charged carboxylate-containing anions, such as those derived from carboxylate-terminated polyamidoamine half-generation dendrimers show charge inversion to give anion charges as high in magnitude as -4, with the degree of adduct formation being inversely related to dendrimer generation. All observations can be interpreted on the basis of charge inversion taking place via a long-lived chemical complex. The lifetime of this complex is related to the strengths and numbers of the interactions of the reactants in the complex. Calculations with model systems are fully consistent with sulfonate groups giving rise to more stable complexes. The kinetic stability of the complex can also be affected by the presence of electrostatic repulsion if it is multiply charged. In general, this situation destabilizes the complex and reduces the likelihood for observation of adducts. The findings highlight the characteristics of multiply charged anions that play roles in determining the nature of charge inversion products associated with protonated peptides and proteins.

The effects of properties of the polypeptide and anionic reactant on the nature of the reaction products are investigated. It is observed that polypeptides composition, sequence, and charge state can play a notable role in determining the distribution of products.

An assessment of three spherical atom-based cutoff approaches such as abrupt truncation, a CHARMM-style electrostatic shift truncation and a force-shift truncation for use with all-atom explicit solvent molecular dynamics (MD) is described. This work demonstrates that by using proper and rigorous techniques, it is possible to correctly model polypeptide dynamics in solution with a spherical cutoff.

Significantly altering the amphipathic structure of a lytic peptide by reshuffling its sequence and/oreplacing a few L-amino acids with their D-enantinomers did not significantly affect the antimicrobial activity of the peptides nor their ability to bind and permeate negatively charged (PE/PG) membranes. This study clearly enables differentiating between the modes of action of an amphipathic alpha-helical lytic peptide, its sequence altered analogues, and their diastereomers.

Ferritins are iron -storage proteins capable of holding up to 4500Fe(Super 3+) ions with in a single water-soluble protein shell made from 24 polypeptide chain. The Glu128Arg/Glu135Arg mutants of Escherichia coli and Rhodobacter capsulatus bacterioferritins are unable to associate into 24-meric structures, with dimmers of polypeptide chains being their stable forms.

Calculations of the diabatic energy surfaces of terminally-blocked amino-acid residues were performed at the Moller-Plesset (MP2) ab initio level of theory with the 6-31G(d,p) basis set. It is essential to have high quality potential-energy surfaces to parametrize a coarse-grain force field, and errors in the parent single-residue all-atom energy surfaces propagate to the mean-field surfaces by numerical integration.

Adsorbed polypeptides such as peripheral proteins disturb the homogeneity of the lipid mixture, which forms membranes. The induced concentration deviations produce an enhanced deviations produce an enhanced adsorption of the polypeptides on the membrane surface and an attractive force between them.

The semisynthesis of the potassium channel KcsA is described. The KcsA polypeptide following chemical ligation was folded into the tetrameric state by incorporation into lipid vesicles and the work described is the first such semisynthesis.