Identification of local variations within secondary structures of proteins

Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method, ASSP (Assignment of Secondary Structure in Proteins), using only the path traversed by the C atoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely -helices, 3(10)-helices, -helices, extended -strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from -helices and extended -strands, 3(10)-helices and -helices were also found to occur in substantial numbers. ASSP was able to discriminate non--helical segments from flanking -helices, which were often identified as part of -helices by other algorithms. ASSP can also lead to the identification of novel SSEs. It is believed that ASSP could provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at .

A cis-Regulatory Mutation in Troponin-I of Drosophila Reveals the Importance of Proper Stoichiometry of Structural Proteins During Muscle Assembly

Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.

Suppression of protein aggregation by gold nanoparticles: a new way to store and transport proteins

Suppression of the aggregation of proteins has tremendous implications in biology and medicine. In the pharmaceuticals industry, aggregation of therapeutically important proteins and peptides while stored, reduces the efficacy and promptness of action leading to, in many instances, intoxication of the patient by the aggregate. Here we report the effect of gold nanoparticles (Au-NPs) in preventing the thermal and chemical aggregation of two unrelated proteins of different size, alcohol dehydrogenase (ADH, 84 kDa) and insulin (6 kDa), respectively, in physiological pH. Our principal observation is that there is a significant reduction (up to 95%) in the extent of aggregation of ADH and insulin in the presence of gold nanoparticles (Au-NPs). Aggregation of these proteins at micromolar concentration is prevented using nanomolar or less amounts of gold nanoparticles which is remarkable since chaperones which prevent such aggregation in vivo are required in micromolar quantity. The prevention of aggregation of these two different proteins under two different denaturing environments has established the role of Au-NPs as a protein aggregation prevention agent. The extent of prevention increases rapidly with the increase in the size of the gold nanoparticles. Protein molecules get physisorbed on the gold nanoparticle surface and thus become inaccessible by the denaturing agent in solution. This adsorption of proteins on AuNPs has been established by a variety of techniques and assays.

Viruses and viral proteins

For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.

Design of symmetric TIM barrel proteins from first principles

Background: Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (alpha/beta)(8) TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts. Methods: Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics. Results: Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a T-m of 44 degrees C and a Gibbs free energy of unfolding (Delta G degrees) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a C-m of 1.6 M and a Delta G degrees of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics. Conclusions: Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

Isotopic fractionation in proteins as a measure of hydrogen bond length

If a deuterated molecule containing strong intramolecular hydrogen bonds is placed in a hydrogenated solvent, it may preferentially exchange deuterium for hydrogen. This preference is due to the difference between the vibrational zero-point energy for hydrogen and deuterium. It is found that the associated fractionation factor (I) is correlated with the strength of the intramolecular hydrogen bonds. This correlation has been used to determine the length of the H-bonds (donor-acceptor separation) in a diverse range of enzymes and has been argued to support the existence of short low-barrier H-bonds. Starting with a potential energy surface based on a simple diabatic state model for H-bonds, we calculate (I) as a function of the proton donor-acceptor distance R. For numerical results, we use a parameterization of the model for symmetric 0-H. ``.0 bonds R. H. McKenzie, Chem. Phys. Lett. 535, 196 (2012)]. We consider the relative contributions of the 0-H stretch vibration, O-H bend vibrations (both in plane and out of plane), tunneling splitting effects at finite temperature, and the secondary geometric isotope effect. We compare our total (I) as a function of R with NMR experimental results for enzymes, and in particular with an earlier model parametrization (D(R), used previously to determine bond lengths. (C) 2015 AIP Publishing LLC.

Evolution of bacterial transcription factors: how proteins take on new tasks, but do not always stop doing the old ones

Many bacterial transcription factors do not behave as per the textbook operon model. We draw on whole genome work, as well as reported diversity across different bacteria, to argue that transcription factors may have evolved from nucleoid-associated proteins. This view would explain a large amount of recent data gleaned from high-throughput sequencing and bioinformatic analyses.

Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold

Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non-redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23-24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co-localized in the cell. Proteins 2015; 83:1766-1786. (c) 2015 Wiley Periodicals, Inc.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-beta Conformation

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-beta(40) (A beta(40)), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a beta-turn, flanked by two beta-sheet regions. We use solid-state NMR data to confirm the presence of the beta-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic A beta fibrils. Significantly, this allows a ``porin'' like beta-barrel structure, providing a structural basis for proposed mechanisms of A beta oligomer toxicity.

Mono layer to Interdigitated Partial Bilayer Smectic C Transition in Thiophene-Based Spacer Mesogens: X-ray Diffraction and C-13 Nuclear Magnetic Resonance Studies

Mesophase organization of molecules built with thiophene at the center and linked via flexible spacers to rigid side arm core units and terminal alkoxy chains has been investigated. Thirty homologues realized by varying the span of the spacers as well as the length of the terminal chains have been studied. In addition to the enantiotropic nematic phase observed for all the mesogens, the increase of the spacer as well as the terminal chain lengths resulted in the smectic C phase. The molecular organization in the smectic phase as investigated by temperature dependent X-ray diffraction measurements revealed an interesting behavior that depended on the length of the spacer vis-a-vis the length of the terminal chain. Thus, a tilted interdigitated partial bilayer organization was observed for molecules with a shorter spacer length, while a tilted monolayer arrangement was observed for those with a longer spacer length. High-resolution solid state C-13 NMR studies carried out for representative mesogens indicated a U-shape for all the molecules, indicating that intermolecular interactions and molecular dynamics rather than molecular shape are responsible for the observed behavior. Models for the mesophase organization have been considered and the results understood in terms of segregation of incompatible parts of the mesogens combined with steric frustration leading to the observed lamellar order.

Rapid NMR Assignments of Proteins by Using Optimized Combinatorial Selective Unlabeling

A new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling is presented. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific assignments, without requiring a contiguously stretch of amino acid residues to be linked, and is applicable to deuterated proteins. We show that a 2D N-15,H-1]HSQC spectrum with two 2D spectra can result in approximate to 50% assignments. The methodology was applied to two proteins: an intrinsically disordered protein (12kDa) and the 29kDa (268 residue) -subunit of Escherichia coli tryptophan synthase, which presents a challenging case with spectral overlaps and missing peaks. The method can augment existing approaches and will be useful for applications such as identifying active-site residues involved in ligand binding, phosphorylation, or protein-protein interactions, even prior to complete resonance assignments.

Electron transfer amongst flavo- and hemo-proteins: diffusible species effect the relay processes, not protein-protein binding

Hitherto, electron transfer (ET) between redox proteins has been deemed to occur via donor-acceptor binding, and diffusible reactive species are considered as deleterious side-products in such systems. Herein, ET from cytochrome P450 reductase (CPR, an animal membrane flavoprotein) and horseradish peroxidase (HRP, a plant hemoprotein) to cytochrome c (Cyt c, a soluble animal hemoprotein) was probed under diverse conditions, using standard assays. ET in the CPR-Cyt c system was critically inhibited by cyanide and sub-equivalent levels of polar one-electron cyclers like copper ions, vitamin C/Trolox and superoxide dismutase. In the presence of lipids, inhibition was also afforded by amphipathic molecules vitamin E, palmitoyl-vitamin C and the membrane hemoprotein, cytochrome b(5). Such nonspecific inhibition (by diverse agents in both aqueous and lipid phases) indicated that electron transfer/relay was effected by small diffusible agents, whose lifetimes are shortened by the diverse radical scavengers. When CPR was retained in a dialysis membrane and Cyt c presented outside in free solution, ET was still observed. Further, HRP (taken at nM levels) catalyzed oxidation of a phenolic substrate was significantly inhibited upon the incorporation of sub-nM levels of Cyt c. The findings imply that CPR-Cyt c or HRP-Cyt c binding is not crucial for ET. Further, fundamental quantitative arguments (based on diffusion/collision) challenge the erstwhile protein-protein binding-assisted ET hypothesis. It is proven beyond reasonable doubt that mobile and diffusible electron carriers (ions and radicals) serve as ``redox-relay agents'' in the biological ET models/setup studied.

Silicon Surface Modification with a Mixed Silanes Layer to Immobilize Proteins for Biosensor with Imaging Ellipsometry

One kind of surface modification method on silicon wafer was presented in this paper. A mixed silanes layer was used to modify silicon surface and rendered the surface medium hydrophobic. The mixed silanes layer contained two kinds of compounds, aminopropyltriethoxysilane (APTES) and methyltriethoxysilane (NITES). A few of APTES molecules in the layer was used to immobilize covalently human immunoglobulin G (IgG) on the silicon surface. The human IgG molecules immobilized covalently on the modified surface could retain their structures well and bind more antibody molecules than that on silicon surface modified with only APTES. This kind of surface modification method effectively improved the sensitivity of the biosensor with imaging ellipsometry.

Copurification of the Lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography.

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.