Identification of beta-barrel membrane proteins based on amino acid composition properties and predicted secondary structure

Unlike all-helices membrane proteins, beta-barrel membrane proteins can not be successfully discriminated from other proteins, especially from all-beta soluble proteins. This paper performs an analysis on the amino acid composition in membrane parts of 12 beta-barrel membrane proteins versus beta-strands of 79 all-beta soluble proteins. The average and variance of the amino acid composition in these two classes are calculated. Amino acids such as Gly, Asn, Val that are most likely associated with classification are selected based on Fishers discriminant ratio. A linear classifier built with these selected amino acids composition in observed beta-strands achieves 100% classification accuracy for 12 membrane proteins and 79 soluble proteins in a four-fold cross-validation experiment. Since at present the accuracy of secondary structure prediction is quite high, a promising method to identify beta-barrel membrane proteins is presented based on the linear classifier coupled with predicted secondary structure. Applied to 241 beta-barrel membrane proteins and 3855 soluble proteins with various structures, the method achieves 85.48% (206/241) sensitivity and 92.53% specificity (3567/3855).     

Molecular simulation of hydrophobin adsorption at an oil-water interface

Hydrophobins are small, amphiphilic proteins expressed by strains of filamentous fungi. They fulfill a number of biological functions, often related to adsorption at hydrophobic interfaces, and have been investigated for a number of applications in materials science and biotechnology. In order to understand the biological function and applications of these proteins, a microscopic picture of the adsorption of these proteins at interfaces is needed. Using molecular dynamics simulations with a chemically detailed coarse-grained potential, the behavior of typical hydrophobins at the water-octane interface is studied. Calculation of the interfacial adsorption strengths indicates that the adsorption is essentially irreversible, with adsorption strengths of the order of 100 k(B)T (comparable to values determined for synthetic nanoparticles but significantly larger than small molecule surfactants and biomolecules). The protein structure at the interface is unchanged at the interface, which is consistent with the biological function of these proteins. Comparison of native proteins with pseudoproteins that consist of uniform particles shows that the surface structure of these proteins has a large effect on the interfacial adsorption strengths, as does the flexibility of the protein.     

Prediction of protein disorder at the domain level

Intrinsically disordered/unstructured proteins exist in a highly flexible conformational state largely devoid of secondary structural elements and tertiary contacts. Despite their lack of a well defined structure, these proteins often fulfill essential regulatory functions. The intrinsic lack of structure confers functional advantages on these proteins, allowing them to adopt multiple conformations and to bind to different binding partners. The structural flexibility of disordered regions hampers efforts solving structures at high resolution by X-ray crystallography and/or NMR. Removing such proteins/regions from high-throughput structural genomics pipelines would be of significant benefit in terms of cost and success rate. In this paper we outline the theoretical background of structural disorder, and review bioinformatic predictors that can be used to delineate regions most likely to be amenable for structure determination. The primary focus of our review is the interpretation of prediction results in a way that enables segmentation of proteins to separate ordered domains from disordered regions.     

Structure, topology, and dynamics of membrane peptides and proteins from solid-state NMR spectroscopy

The high-resolution structure of membrane proteins is notoriously difficult to determine due to the hydrophobic nature of the protein-membrane complexes. Solid-state NMR spectroscopy is a unique and powerful atomic-resolution probe of the structure and dynamics of these important biological molecules. A number of new solid-state NMR methods for determining the depth of insertion, orientation, oligomeric structure, and long-range (10-15 A) distances of membrane proteins are summarized. Membrane protein depths can now be determined using several complementary techniques with varying site-specificity, distance precision, and mobility requirement on the protein. Membrane protein orientation can now be determined with or without macroscopic alignment, the latter providing a novel alternative for orientation determination of intrinsically curvature-inducing proteins. The novel analyses of beta-sheet membrane protein orientation are described. The quaternary structure of membrane peptide assemblies can now be elucidated using a 19F spin diffusion technique that simultaneously yields the oligomeric number and intermolecular distances up to 15 A. Finally, long-range distances up to approximately 10 A can now be measured using 1H spins with an accuracy of better than 1 A. These methods are demonstrated on several beta-sheet membrane peptides with antimicrobial activities and on two alpha-helical ion-channel proteins. Finally, we show that the nearly ubiquitous dynamics of membrane proteins can be readily examined using 2D correlation experiments. An intimate appreciation of molecular motion in these systems not only leads to important insights into the specific function of these membrane proteins but also may be exploited for other purposes such as orientation determination.     

Identification of β-barrel membrane proteins based on amino acid composition properties and predicted secondary structure

Unlike all-helices membrane proteins, β-barrel membrane proteins can not be successfully discriminated from other proteins, especially from all-β soluble proteins. This paper performs an analysis on the amino acid composition in membrane parts of 12 β-barrel membrane proteins versus β-strands of 79 all-β soluble proteins. The average and variance of the amino acid composition in these two classes are calculated. Amino acids such as Gly, Asn, Val that are most likely associated with classification are selected based on Fishers discriminant ratio. A linear classifier built with these selected amino acids composition in observed β-strands achieves 100% classification accuracy for 12 membrane proteins and 79 soluble proteins in a four-fold cross-validation experiment. Since at present the accuracy of secondary structure prediction is quite high, a promising method to identify β-barrel membrane proteins is presented based on the linear classifier coupled with predicted secondary structure. Applied to 241 β-barrel membrane proteins and 3855 soluble proteins with various structures, the method achieves 85.48% (206/241) sensitivity and 92.53% specificity (3567/3855).     

Glycosylation of the Arg-gingipains of Porphyromonas gingivalis and comparison with glycoconjugate structure and synthesis in other bacteria

Post-translational modification of proteins by covalent attachment of sugars to the protein backbone (protein glycosylation) is the most common post-translational modification in the eucaryotic cell. However, the addition of carbohydrates to proteins of Eubacteria and Archaea has been demonstrated and accepted only recently. There is now a rapidly expanding list of bacterial glycoproteins that have been characterised from a variety of different organisms including many important pathogens. The Arg-gingipains of Porphyromonas gingivalis are recent additions to this list. In this review we present a summary of our investigations on the structure of the glycan additions to these proteolytic enzymes, the genetics of the glycosylation process and some of the effects on enzyme function and recognition. These findings are placed in the context of the current status of understanding of glycoconjugate structure and synthesis in other bacteria. Given the importance of glycosylation of eucaryotic proteins to their stability, structure, resistance to proteolysis and recognition, the modifications to the proteases described in the present report are likely to have a functional role in the properties of these enzymes in periodontal disease.     

Mapping the electronic structure of the blue copper site in plastocyanin by NMR relaxation

The biological function of metalloproteins stems from the electronic and geometric structures of their active sites. Thus, in blue copper proteins such as plastocyanins, an unusual electronic structure of the metal site is believed to contribute to the rapid, long-range electron-transfer reactivity that characterizes these proteins. To clarify this structure-function relationship, numerous quantum chemical calculations of the electronic structure of the blue copper proteins have been made. However, the obtained structures depend strongly on the applied model. Experimental approaches based on ENDOR spectroscopy and X-ray absorption have also been used to elucidate the electronic structure of the blue copper site. Still, the determination of the electronic structure relies on a calibration with quantum chemical calculations, performed on small model complexes. Here we present an approach that allows a direct experimental mapping of the electron spin delocalization in paramagnetic metalloproteins using oxidized plastocyanin from Anabaena variabilis as an example. The approach utilizes the longitudinal paramagnetic relaxation of protons close to the metal site and relies on the dependence of these relaxations on the spatial distribution of the unpaired electron of the metal ion. Surprisingly it is found that the unpaired electron of the copper ion in plastocyanin is less delocalized than predicted by most of the quantum chemical calculations.     

Production and crystallization of protein domains: how useful are disorder predictions ?

The failure to produce and/or crystallize proteins is often due to their modular structure. There exists therefore considerable interest to develop strategies for tailoring proteins into crystallizable domains. In the framework of a Structural Genomics Project on soluble yeast proteins, we have tested the expression of numerous genetic constructs of our targets in order to produce and crystallize proteins and protein domains and solve their three-dimensional structure. In some cases, the choice of the domain boundaries was guided by prediction from sequence using various software packages, including Prelink, a home-made prediction method for detecting unfolded regions. In other cases, large numbers of constructs were generated using molecular biology or biochemical methods. In this paper, we analyze the results of the over-expression in E. coli and crystallization of these constructs, and compare these with the predictions that can be obtained from our software and from others.     

Circular and linear dichroism of proteins

Circular dichroism (CD) is an important technique in the structural characterisation of proteins, and especially for secondary structure determination. The CD of proteins can be calculated from first principles using the so-called matrix method, with an accuracy which is almost quantitative for helical proteins. Thus, for proteins of unknown structure, CD calculations and experimental data can be used in conjunction to aid structure analysis. Linear dichroism (LD) can be calculated using analogous methodology and has been used to establish the relative orientations of subunits in proteins and protein orientation in an environment such as a membrane. However, simple analysis of LD data is not possible, due to overlapping transitions. So coupling the calculations and experiment is an important strategy. In this paper, the use of LD for the determination of protein orientation and how these data can be interpreted with the aid of calculations, are discussed. We review methods for the calculation of CD spectra, focusing on semiempirical and ab initio parameter sets used in the matrix method. Lastly, a new web interface for online CD and LD calculation is presented.     

Structure, Stability, and Activity of Adsorbed Enzymes

A proteolytic enzyme, α-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solution onto a hydrophobic Teflon surface and a hydrophilic silica surface. We investigated the influence of adsorption on the structure, the structure thermal stability and the activity of these enzymes. Probing the protein structure by circular dichroism spectroscopy indicates that Teflon promotes the formation of helical structure in α-chymotrypsin, but the reverse effect is found with cutinase. The perturbed protein structures on Teflon are remarkably stable, showing no heat-induced structural transitions up to 100°C, as monitored by differential scanning calorimetry. Contact with the hydrophilic silica surface leads to a loss in the helix content of both proteins. Differential scanning calorimetry points to a heterogeneous population of adsorbed protein molecules with respect to their conformational states. The fraction of the native-like conformation in the adsorbed layer increases with increasing coverage of the silica surface by the proteins. The specific enzymatic activity in the adsorbed state qualitatively correlates with the fraction of proteins in the native-like conformation.     

Chemical and semisynthesis of posttranslationally modified proteins

Posttranslational modifications of proteins play crucial roles in health and disease by affecting numerous aspects of protein structure, function, stability and sub cellular localization. Yet understanding the effects of these modifications on several of these processes at the molecular level has been hindered by the lack of homogeneously modified proteins obtained via traditional biochemical and molecular biology approaches. Moreover, the preparation of such bioconjugates at a workable level is highly demanding. Recent advances in protein chemistry applying chemical and semisynthetic approaches are becoming increasingly beneficial to overcome these challenges. These methods allow site-specific modifications of a desired protein and afford the product in large quantities for biochemical and structural analyses. In this review, we survey these efforts and their importance in dissecting the role of several posttranslational modifications in various proteins. Several examples are presented where glycosylated, phosphorylated, ubiquitinated, lipidated, acetylated and methylated proteins were prepared.     

Protein folding at emulsion oil/water interfaces

It has long been known that proteins change their conformation upon adsorption to emulsion oil/water interfaces. However, it is only recently that details of the specifics of these structural changes have emerged. The development of synchrotron radiation circular dichroism (SRCD), combined with advances in FTIR spectroscopy, has allowed the secondary and tertiary structure of proteins adsorbed at emulsion oil/water interfaces to be studied. SRCD in particular has provided quantitative information and has enabled new insights into the mechanisms and forces driving protein structure re-arrangement to be achieved.The extent of conformational re-arrangement of proteins at emulsion interfaces is influenced by several factors including; the inherit flexibility of the protein, the distribution of hydrophobic/hydrophilic domains within the protein sequence and the hydrophobicity of the oil phase. In general, proteins lose much of their tertiary structure upon adsorption to the oil/water interface and have considerable amounts of non-native secondary structure. Two key conformations have been identified in the structure of proteins at interfaces, intermolecular β-sheet and α-helix. The preferred conformation appears to be the α-helix which is the most compact amphipathic conformation at the oil/water interface. The polarity of the oil phase can have a considerable influence on the degree of protein conformational re-arrangement because it acts as a solvent for hydrophobic amino acids. The new conformation of proteins at interfaces also means that proteins undergo less heat induced re-arrangement at interfaces than in solution. Different conformations of proteins at interfaces impact on emulsification capability, emulsion stability and protein/emulsion digestion. Hence advances in the understanding of protein conformation at interfaces can help to identify suitable proteins and conditions for the preparation of emulsion based food products.     

Green and red fluorescent proteins: photo- and thermally induced dynamics probed by site-selective spectroscopy and hole burning.

The cloning and expression of autofluorescent proteins in living matter, combined with modern imaging techniques, have thoroughly changed the world of bioscience. In particular, such proteins are widely used as genetically encoded labels to track the movement of proteins as reporters of cellular signals and to study protein-protein interactions by fluorescence resonance energy transfer (FRET). Their optical properties, however, are complex and it is important to understand these for the correct interpretation of imaging data and for the design of new fluorescent mutants. In this Minireview we start with a short survey of the field and then focus on the photo- and thermally induced dynamics of green and red fluorescent proteins. In particular, we show how fluorescence line narrowing and high-resolution spectral hole burning at low temperatures can be used to unravel the photophysics and photochemistry and shed light on the intricate electronic structure of these proteins.     

The formation of amyloid fibrils from proteins in the lysozyme family

Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. More recently, these fibrils have been shown to be useful as structural scaffolds in both natural biological systems and nanotechnology applications. The intense interest in amyloid fibrils has led to the investigation of well-characterized proteins, such as hen egg white lysozyme (HEWL), as model systems to examine structural and mechanistic principles that may be generally applicable to all amyloid fibrils. The purpose of this review is to critically examine the fibril-formation literature of proteins in the lysozyme family with respect to the known structure and folding properties of these proteins. The goal is to identify similarities and differences within the family, examine general misfolding / aggregation principles, and identify key areas of importance for future work on the fibril formation of these proteins.     

Using the telobox to search for plant telomere binding proteins

Telobox is a Myb-related DNA-binding domain which is present in a number of yeast, plant and animal proteins. Its capacity to bind preferentially double-stranded telomeric DNA has been used in numerous studies to search for candidate telomeric proteins in various organisms, including plants. Here we provide an overview of these studies with a special emphasis on plants, where a specific subfamily of the proteins possessing the N-terminally positioned telobox is present in addition to more common C-terminal telobox proteins. We further demonstrate the presence of a telobox protein (CpTBP1) in Cestrum parqui, a plant lacking typical telomeres and telomerase. The protein shows nuclear localisation and association with chromatin. The role of this protein in ancestral and current telomere structure is discussed in the evolutionary context. Altogether, the present overview shows the importance of the telobox domain in a search for candidate telomere proteins but at the same time warns against oversimplified identification of any telobox protein with telomere structure without appropriate evidence of its telomeric localisation and function.     

Decharging of globular proteins and protein complexes in electrospray

Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.     

Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry

Protein adsorption behavior is at the heart of many of today's research fields including biotechnology and materials science. With understanding of protein-surface interactions, control over the conformation and orientation of immobilized species may ultimately allow tailor-made surfaces to be generated. In this contribution protein-surface interactions have been examined with particular focus on surface curvature with and without surface chemistry effects. Silica spheres with diameters in the range 15-165 nm with both hydrophilic and hydrophobic surface chemistries have been used as model substrates. Two proteins differing in size and shape, bovine serum albumin (BSA) and bovine fibrinogen (Fg), have been used in model studies of protein binding with detailed secondary structure analysis being performed using infrared spectroscopy (IR) on surface-bound proteins. Although trends in binding affinity and saturation values were similar for both proteins, albumin is increasingly less ordered on larger substrates, while fibrinogen, in contrast, loses secondary structure to a greater extent when adsorbing onto particles with high surface curvature. These effects are compounded by surface chemistry, with both proteins becoming more denatured on hydrophobic surfaces. Both surface chemistry and topography play key roles in determining the structure of the bound proteins. A model of the binding characteristics of these two proteins onto surfaces having differing curvature and chemistry is presented. We propose that properties of an adsorbed protein layer may be guided through careful consideration of surface structure, allowing the fabrication of materials/surface coatings with tailored bioactivity.     

Cysteine and histidine shuffling: mixing and matching cysteine and histidine residues in zinc finger proteins to afford different folds and function

Zinc finger proteins utilize zinc for structural purposes: zinc binds to a combination of cysteine and histidine ligands in a tetrahedral coordination geometry facilitating protein folding and function. While much is known about the classical zinc finger proteins, which utilize a Cys(2)His(2) ligand set to coordinate zinc and fold into an anti-parallel beta sheet/alpha helical fold, there are thirteen other families of 'non-classical' zinc finger proteins for which relationships between metal coordination and protein structure/function are less defined. This 'Perspective' article focuses on two classes of these non-classical zinc finger proteins: Cys(3)His type zinc finger proteins and Cys(2)His(2)Cys type zinc finger proteins. These proteins bind zinc in a tetrahedral geometry, like the classical zinc finger proteins, yet they adopt completely different folds and target different oligonucleotides. Our current understanding of the relationships between ligand set, metal ion, fold and function for these non-classical zinc fingers is discussed.     

Ab initio study of the effect of side-chain reactions on the electronic structure of proteins

The effect of side-chain protonation and tautomerization of histidine on the electronic structure of proteins is investigated. Ab initio band structure results for protonated polyhistidine, polyhistidine in both tautomeric forms, polyglycine and polyalanine are reported. The density of states of the corresponding composite aperiodic polypeptides is studied using the simple negative factor counting method. It is shown how these side-chain reactions reduce the fundamental gap in aperiodic proteins, thereby modifying their conduction properties.     

Correlation of the side-chain hubs with the functional residues in DNA binding protein structures

The structure networks of DNA-binding proteins have been constructed and analyzed. The detailed analysis of the networks indicates a strong relation between the positions of the residues interacting with DNA and those that form extensive interactions within the protein structure (called hubs). This study shows that the functional residues in these proteins are held in place by efficient scaffolding of the structure using side-chain interactions, thus highlighting the role of these side-chain hubs with respect to the functional residues in the protein structure.