Characterization of dynamic behavior of side groups of globular proteins by spin-label, NMR and luminescence polarization methods

In this work we suggest a quantitative estimation of a complicated motion of side groups of globular proteins. In the general case, three basic parameters determine the motion: (a) rotational correlation time of a side unit under study, or covalently bound spin label, or dye, (b) parameter S that reflects sterical restrictions for re-orientation of the given unit (these two parameters depending on the side-chain structure and its conformational change within the immediate dynamic protein surrounding whereas correlation times of side units on microviscosity in addition), (c) rotational correlation time of protein globule. These parameters can be measured by spin-label, NMR and fluorescence polarization techniques. An attempt to describe a complicated dynamic behaviour of side units of protein macromolecules with a single dynamics parameter--rotational correlation time--not only leads to a loss of part of information about the local structural dynamics of macromolecules but also can diminish the tau value.     

Analysis of tryptophan surface accessibility in proteins by MALDI-TOF mass spectrometry

Surface accessible amino acids can play an important role in proteins. They can participate in enzyme's active center structure or in specific intermolecular interactions. Thus, the information about selected amino acids' surface accessibility can contribute to the understanding of protein structure and function. In this paper, we present a simple method for surface accessibility mapping of tryptophan side chains by their chemical modification and identification by MALDI-TOF mass spectrometry. The reaction with 2-hydroxy-5-nitrobenzyl bromide, a common and highly specific covalent modification of tryptophan, seems to be very useful for this purpose. The method was tested on four model proteins with known spatial structure. In the native proteins (1) only surface accessible tryptophan side chains were found to react with the modification agent and (2) no buried one was found to react at lower reagent concentrations. These results indicate that the described method can be a potent tool for identification of surface-located tryptophan side chain in a protein of unknown conformation.     

Angle determination for side views in single particle electron microscopy

In order to build a first model in single particle electron microscopy the relative angular orientation of each image of a protein complex must be determined. These orientations can be described by three Eulerian angles. Images of complexes that present the same view can be aligned in two-dimensions and averaged in order to increase their signal-to-noise ratio. Based on these averaged images, several standard approaches exist for determining Euler angles for randomly oriented projection images. The common lines and angular reconstitution methods work well for particles with symmetry while the random conical tilting and related orthogonal tilt reconstruction methods work in most cases but require the acquisition of tilt pairs of images. For the situation where views of particles can be identified that are rotations about a single axis parallel to the grid, an alternative algorithm to determine the orientations of class averages without the need to acquire tilt pairs can be applied. This type of view of a complex is usually called a side view. This paper describes the detailed workings and characterization of an algorithm, named rotational analysis, which uses real-space fiducial markers derived from the averages themselves to determine the Euler angles for side views. We demonstrate how this algorithm works in practice by applying it to a data set of images of affinity-purified bovine mitochondrial ATP synthase.     

Protein folding at single-molecule resolution

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation-{pi} interactions

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation-pi interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second-derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N-acetylated C-ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation-pi interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.     

Competing views on cancer

Despite intense research efforts that have provided enormous insight, cancer continues to be a poorly understood disease. There has been much debate over whether the cancerous state can be said to originate in a single cell or whether it is a reflection of aberrant behaviour on the part of a ‘society of cells’. This article presents, in the form of a debate conducted among the authors, three views of how the problem might be addressed. We do not claim that the views exhaust all possibilities. These views are (a) the tissue organization field theory (TOFT) that is based on a breakdown of tissue organization involving many cells from different embryological layers, (b) the cancer stem cell (CSC) hypothesis that focuses on genetic and epigenetic changes that take place within single cells, and (c) the proposition that rewiring of the cell’s protein interaction networks mediated by intrinsically disordered proteins (IDPs) drives the tumorigenic process. The views are based on different philosophical approaches. In detail, they differ on some points and agree on others. It is left to the reader to decide whether one approach to understanding cancer appears more promising than the other.     

Lipid–protein interactions in biological membranes: A dynamic perspective

Though an increasing number of biological functions at the membrane are attributed to direct associations between lipid head groups and protein side chains or lipid protein hydrophobic attractive forces, surprisingly limited information is available about the dynamics of these interactions. The static in vitro representation provided by membrane protein structures, including very insightful lipid–protein binding geometries, still fails to recapitulate the dynamic behavior characteristic of lipid membranes. Experimental measures of the interaction time of lipid–protein association are very rare, and have only provided order-of-magnitude estimates in an extremely limited number of systems. In this review, a brief outline of the experimental approaches taken in this area to date is given. The bulk of the review will focus on two methods that are promising techniques for measuring lipid–protein interactions: time-resolved fluorescence microscopy, and two-dimensional infrared (2D IR) spectroscopy. Time-resolved fluorescence microscopy is the name given to a sophisticated toolbox of measurements taken using pulsed laser excitation and time-correlated single photon counting (TCSPC). With this technique the dynamics of interaction can be measured on the time scale of nanoseconds to milliseconds. 2D IR is a femtosecond nonlinear spectroscopy that can resolve vibrational coupling between lipids and proteins at molecular-scale distances and at time scales from femtoseconds to picoseconds. These two methods are poised to make significant advances in our understanding of the dynamic properties of biological membranes. This article is part of a Special Issue entitled: Membrane protein structure and function.     

A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics.

A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild-type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of the wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 "DTT-sensitive" mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the native wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated at most sites without completely preventing folding. Because this genetic screen is based on changes in folding energetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.     

Some quantitative characteristics of the frog retinal rod outer segments]

Retinal rod outer segments in frogs were studied by means of light microscopy, refractometry, microspectrophotometry, and electron microscopy. Analysis of the data obtained shows that an unidentified substance, which makes up about 50% of outer segment dry weight, is lost during routine biochemical investigations. The protein parts of the rhodopsin molecules make up 85% of the outer segments proteins and 25% of outer segment dry weight. Rhodopsin molecules can be arranged in a square array with a unit cell side of about 7 nm on one side of each disk membrane. Lipids in a single membrane occupy only 2 nm, and disk membranes are strongly hydrated.     

Fluorescence characterization of denatured proteins

Characterization of unfolded states, while critical to a complete understanding of protein folding, is inherently difficult due to structural heterogeneity and dynamic interchange between states. The growing body of work focusing on single molecule fluorescence techniques for the study of protein folding, also highlights their potential for studies of unfolded proteins. These methods can obtain conformational information about individual subpopulations of molecules in an ensemble, and measure dynamics without the need for synchronization. The studies highlighted here demonstrate the promise of these techniques for obtaining novel information about unfolded states in vitro and in more physiologically relevant milieu.     

PRIME: automatically extracted PRotein Interactions and Molecular Information databasE

With the exponentially increasing amount of information in the biomedical field, the significance of advanced information retrieval and information extraction, as well as the role of databases, has been increasing. PRIME is an integrated gene/protein informatics database based on natural language processing. It provides automatically extracted protein/family/gene/compound interaction information including both physical and genetic interactions, gene ontology based functions, and graphic pathway viewers. Gene/protein/family names and functional terms are recognized based on dictionaries developed in our laboratory. The interaction and functional information are extracted by syntactic dependencies and various phrase patterns. We have included about 920,000 (non-redundant) protein interactions and 360,000 annotated gene-function relationships for major eukaryotes. By combining the sequence and text information, the pathway comparison between two organisms and simple pathway deduction based on other organism interaction data, and pathway filtering using tissue expression data, are also available. This database is accessible at http://prime.ontology.ims.u-tokyo.ac.jp:8081.     

Side-chain interactions determine amyloid formation by model polyglutamine peptides in molecular dynamics simulations

The pathological manifestation of nine hereditary neurodegenerative diseases is the presence within the brain of aggregates of disease-specific proteins that contain polyglutamine tracts longer than a critical length. To improve our understanding of the processes by which polyglutamine-containing proteins misfold and aggregate, we have conducted molecular dynamics simulations of the aggregation of model polyglutamine peptides. This work was accomplished by extending the PRIME model to polyglutamine. PRIME is an off-lattice, unbiased, intermediate-resolution protein model based on an amino acid representation of between three and seven united atoms, depending on the residue being modeled. The effects of hydrophobicity on the system are studied by varying the strength of the hydrophobic interaction from 12.5% to 5% of the hydrogen-bonding interaction strength. In our simulations, we observe the spontaneous formation of aggregates and annular structures that are made up of beta-sheets starting from random configurations of random coils. This result was interesting because tubular protofibrils were recently found in experiments on polyglutamine aggregation and because of Perutz's prediction that polyglutamine would form water-filled nanotubes.     

Bioinformatic assessment of mass spectrometric chemical derivatisation techniques for proteome database searching

Identification of proteins from the mass spectra of peptide fragments generated by proteolytic cleavage using database searching has become one of the most powerful techniques in proteome science, capable of rapid and efficient protein identification. Using computer simulation, we have studied how the application of chemical derivatisation techniques may improve the efficiency of protein identification from mass spectrometric data. These approaches enhance ion yield and lead to the promotion of specific ions and fragments, yielding additional database search information. The impact of three alternative techniques has been assessed by searching representative proteome databases for both single proteins and simple protein mixtures. For example, by reliably promoting fragmentation of singly-charged peptide ions at aspartic acid residues after homoarginine derivatisation, 82% of yeast proteins can be unambiguously identified from a single typical peptide-mass datum, with a measured mass accuracy of 50 ppm, by using the associated secondary ion data. The extra search information also provides a means to confidently identify proteins in protein mixtures where only limited data are available. Furthermore, the inclusion of limited sequence information for the peptides can compensate and exceed the search efficiency available via high accuracy searches of around 5 ppm, suggesting that this is a potentially useful approach for simple protein mixtures routinely obtained from two-dimensional gels.     

A unified mechanism for protein folding: predetermined pathways with optional errors

There is a fundamental conflict between two different views of how proteins fold. Kinetic experiments and theoretical calculations are often interpreted in terms of different population fractions folding through different intermediates in independent unrelated pathways (IUP model). However, detailed structural information indicates that all of the protein population folds through a sequence of intermediates predetermined by the foldon substructure of the target protein and a sequential stabilization principle. These contrary views can be resolved by a predetermined pathway--optional error (PPOE) hypothesis. The hypothesis is that any pathway intermediate can incorporate a chance misfolding error that blocks folding and must be reversed for productive folding to continue. Different fractions of the protein population will then block at different steps, populate different intermediates, and fold at different rates, giving the appearance of multiple unrelated pathways. A test of the hypothesis matches the two models against extensive kinetic folding results for hen lysozyme which have been widely cited in support of independent parallel pathways. The PPOE model succeeds with fewer fitting constants. The fitted PPOE reaction scheme leads to known folding behavior, whereas the IUP properties are contradicted by experiment. The appearance of a conflict with multipath theoretical models seems to be due to their different focus, namely on multitrack microscopic behavior versus cooperative macroscopic behavior. The integration of three well-documented principles in the PPOE model (cooperative foldons, sequential stabilization, optional errors) provides a unifying explanation for how proteins fold and why they fold in that way.     

Comparison of the folding mechanism of highly homologous proteins in the lipid‐binding protein family

The folding mechanism of two closely related proteins in the intracellular lipid‐binding protein family, human bile acid‐binding protein (hBABP), and rat bile acid‐binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence. Both of these single domain proteins fit well to a two‐state model for unfolding by fluorescence and circular dichroism at equilibrium. Three phases were observed during the unfolding of rBABP by fluorescence but only one phase was observed during the unfolding of hBABP, suggesting that at least two kinetic intermediates accumulate during the unfolding of rBABP that are not observed during the unfolding of hBABP. Fluorine NMR was used to examine the equilibrium unfolding behavior of the W49 side chain in 6‐fluorotryptophan‐labeled rBABP and hBABP. The structure of rBABP appears to be more dynamic than that of hBABP in the vicinity of W49 in the absence of denaturant, and urea has a greater effect on this dynamic behavior for rBABP than for hBABP. As such, the folding behavior of highly sequence related proteins in this family can be quite different. These differences imply that moderately sized proteins with high sequence and structural similarity can still populate quite different structures during folding. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Integration of genome data and protein structures: prediction of protein folds, protein interactions and "molecular phenotypes" of single nucleotide polymorphisms

With the massive amount of sequence and structural data being produced, new avenues emerge for exploiting the information therein for applications in several fields. Fold distributions can be mapped onto entire genomes to learn about the nature of the protein universe and many of the interactions between proteins can now be predicted solely on the basis of the genomic context of their genes. Furthermore, by utilising the new incoming data on single nucleotide polymorphisms by mapping them onto three-dimensional structures of proteins, problems concerning population, medical and evolutionary genetics can be addressed.     

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

ABSTRACT: BACKGROUND: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. RESULTS: Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3[PRIME] poly(A) sequence identifying the 3[PRIME] end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. CONCLUSIONS: NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.     

The interplay between transient α-helix formation and side chain rotamer distributions in disordered proteins probed by methyl chemical shifts

The peptide backbones of disordered proteins are routinely characterized by NMR with respect to transient structure and dynamics. Little experimental information is, however, available about the side chain conformations and how structure in the backbone affects the side chains. Methyl chemical shifts can in principle report the conformations of aliphatic side chains in disordered proteins and in order to examine this two model systems were chosen: the acid denatured state of acyl-CoA binding protein (ACBP) and the intrinsically disordered activation domain of the activator for thyroid hormone and retinoid receptors (ACTR). We find that small differences in the methyl carbon chemical shifts due to the γ-gauche effect may provide information about the side chain rotamer distributions. However, the effects of neighboring residues on the methyl group chemical shifts obscure the direct observation of γ-gauche effect. To overcome this, we reference the chemical shifts to those in a more disordered state resulting in residue specific random coil chemical shifts. The (13)C secondary chemical shifts of the methyl groups of valine, leucine, and isoleucine show sequence specific effects, which allow a quantitative analysis of the ensemble of χ(2)-angles of especially leucine residues in disordered proteins. The changes in the rotamer distributions upon denaturation correlate to the changes upon helix induction by the co-solvent trifluoroethanol, suggesting that the side chain conformers are directly or indirectly related to formation of transient α-helices.