Prediction of amyloidogenic and disordered regions in protein chains

The determination of factors that influence protein conformational changes is very important for the identification of potentially amyloidogenic and disordered regions in polypeptide chains. In our work we introduce a new parameter, mean packing density, to detect both amyloidogenic and disordered regions in a protein sequence. It has been shown that regions with strong expected packing density are responsible for amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for eight of 12 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Our findings support the concept that the mechanism of amyloid fibril formation is similar for different peptides and proteins. Moreover, we have demonstrated that regions with weak expected packing density are responsible for the appearance of disordered regions. Our method has been tested on datasets of globular proteins and long disordered protein segments, and it shows improved performance over other widely used methods. Thus, we demonstrate that the expected packing density is a useful value with which one can predict both intrinsically disordered and amyloidogenic regions of a protein based on sequence alone. Our results are important for understanding the structural characteristics of protein folding and misfolding.     

The analysis of protein folding kinetic data produced in protein engineering experiments

Over the past decade, the "protein engineering method" has been used to investigate the folding pathways of more than 20 different proteins. This method involves measuring the folding and unfolding rates of mutant proteins with single amino acid substitutions spread across the sequence. Comparison of folding rates of the mutant proteins to that of the wild-type protein allows the calculation of the phi value, which can be used to evaluate the stabilizing contribution of an amino acid side chain to the structure of the folding transition state. Here, we review the methodology for analysing data collected in protein engineering folding kinetics studies. We discuss the calculation of folding rates and kinetic m values, the estimation of errors in folding kinetics experiments, phi value calculation including potential pitfalls of the analysis, Br?nsted plots, detecting Hammond behaviour, and the analysis of curved chevron plots.     

Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA.

1. The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. 2. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26 000 was obtained. 3. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated.     

Characterization of a novel protein inhibitor of protein kinases specific to acidic ribosomal proteins

The ribosomal stalk composed of acidic P1/P2 proteins and protein P0 is involved directly in the interaction of the elongation factors and mRNAs with the ribosome during protein synthesis. All P proteins are found to be phosphorylated in eucaryotic organisms. In Saccharomyces cerevisiae five different cAMP-independent protein kinases phosphorylating P proteins have been identified and characterized. In contrast to many other protein kinases, relatively little is known about inhibitors of these enzymes. A new protein inhibitor of protein kinases has been purified and characterized. It is a small (18.5 kDa) and acidic (pI = 4.2) protein with high inhibitory potency for PK60S and CK 2. The inhibitor is competitive with respect to protein substrates with Ki values in the range of approximately 6.5 microM for PK60S and approximately 22 microM for CK 2.     

Proteomic applications of protein quantification by isotope-dilution mass spectrometry

Over the decades, isotope-dilution mass spectrometry (IDMS) has been implemented extensively for accurate quantification of drugs, metabolites and peptides in body fluids and tissues. More recently, it has been extended for quantifying specific proteins in complex mixtures. In this extended methodology, proteins are subjected to endoprotease action and specific resultant peptides are quantified by using synthetic stable isotope-labeled standard (SIS) peptides and IDMS. This article outlines the utilities and applications of quantifying proteins by IDMS, emphasizing its complementary value to global survey-based proteomic studies. The potential of SIS peptides to provide quantitative insights into cell signaling is also highlighted, with specific examples. Finally, we propose several novel mass spectrometric data acquisition strategies for large-scale applications of IDMS and SIS peptides in systems biology and protein biomarker validation studies.     

Proteomic applications of protein quantification by isotope-dilution mass spectrometry

Over the decades, isotope-dilution mass spectrometry (IDMS) has been implemented extensively for accurate quantification of drugs, metabolites and peptides in body fluids and tissues. More recently, it has been extended for quantifying specific proteins in complex mixtures. In this extended methodology, proteins are subjected to endoprotease action and specific resultant peptides are quantified by using synthetic stable isotope-labeled standard (SIS) peptides and IDMS. This article outlines the utilities and applications of quantifying proteins by IDMS, emphasizing its complementary value to global survey-based proteomic studies. The potential of SIS peptides to provide quantitative insights into cell signaling is also highlighted, with specific examples. Finally, we propose several novel mass spectrometric data acquisition strategies for large-scale applications of IDMS and SIS peptides in systems biology and protein biomarker validation studies.     

New insights into the mechanisms of protein palmitoylation

Since its discovery more than 30 years ago, protein palmitoylation has been shown to have a role in protein-membrane interactions, protein trafficking, and enzyme activity. Until recently, however, the molecular machinery that carries out reversible palmitoylation of proteins has been elusive. In fact, both enzymatic and nonenzymatic S-acylation reaction mechanisms have been proposed. Recent reports of protein palmitoyltransferases in Saccharomyces cerevisiae and Drosophila provide the first glimpse of enzymes that carry out protein palmitoylation. Equally important is the mechanism of depalmitoylation. Two major classes of protein palmitoylthioesterases have been described. One family is lysosomal and is involved in protein degradation. The second is cytosolic and removes palmitoyl moieties preferentially from proteins associated with membranes. This review discusses recent advances in the understanding of mechanisms of addition of palmitate to proteins and removal of palmitate from proteins.     

Benchmarking protein–protein interface predictions: Why you should care about protein size

A number of predictive methods have been developed to predict protein–protein binding sites. Each new method is traditionally benchmarked using sets of protein structures of various sizes, and global statistics are used to assess the quality of the prediction. Little attention has been paid to the potential bias due to protein size on these statistics. Indeed, small proteins involve proportionally more residues at interfaces than large ones. If a predictive method is biased toward small proteins, this can lead to an over‐estimation of its performance. Here, we investigate the bias due to the size effect when benchmarking protein‐protein interface prediction on the widely used docking benchmark 4.0. First, we simulate random scores that favor small proteins over large ones. Instead of the 0.5 AUC (Area Under the Curve) value expected by chance, these biased scores result in an AUC equal to 0.6 using hypergeometric distributions, and up to 0.65 using constant scores. We then use real prediction results to illustrate how to detect the size bias by shuffling, and subsequently correct it using a simple conversion of the scores into normalized ranks. In addition, we investigate the scores produced by eight published methods and show that they are all affected by the size effect, which can change their relative ranking. The size effect also has an impact on linear combination scores by modifying the relative contributions of each method. In the future, systematic corrections should be applied when benchmarking predictive methods using data sets with mixed protein sizes. Proteins 2014; 82:1444–1452. © 2014 Wiley Periodicals, Inc.     

Chemical protein synthesis

Since the mid-1990s, chemical synthesis has emerged as a powerful technique for the study of structure/function relationships in proteins. During the review period, the applicability of chemical protein synthesis techniques has been significantly broadened by increases in the size of synthetically accessible proteins through two new techniques: solid-phase protein synthesis and expressed protein ligation. Also in the period under review, synthetic access to novel classes of proteins has been established, including metalloproteins with tuned properties and integral membrane proteins.     

Computational protein design promises to revolutionize protein engineering

Natural evolution has produced an astounding array of proteins that perform the physical and chemical functions required for life on Earth. Although proteins can be reengineered to provide altered or novel functions, the utility of this approach is limited by the difficulty of identifying protein sequences that display the desired properties. Recently, advances in the field of computational protein design (CPD) have shown that molecular simulation can help to predict sequences with new and improved functions. In the past few years, CPD has been used to design protein variants with optimized specificity of binding to DNA, small molecules, peptides, and other proteins. Initial successes in enzyme design highlight CPD's unique ability to design function de novo. The use of CPD for the engineering of potential therapeutic agents has demonstrated its strength in real-life applications.     

Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT)

In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (MudPIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture.     

Protein adsorption at solid-liquid interfaces: Part II--Adsorption from binary protein mixture

Extent of adsorption of proteins at alumina-water interface from solutions containing binary mixture of beta-lactoglobulin and bovine serum albumin (BSA), beta-lactoglobulin and gelatin, and gelatin and bovine serum albumin has been estimated as functions of protein concentrations at varying pH, ionic strength, temperature and weight fraction ratios of protein mixture. The extent of adsorption (gamma lacw) of lactoglobulin in the presence of BSA increases with increase of protein concentration (Clac) until it reaches a maximum but a fixed value gamma lacw(m). Extent of adsorption gamma serw also initially increases with increase of protein concentrations until it reaches maximum value gamma serw(m). Beyond these protein concentrations, adsorbed BSA is gradually desorbed due to the preferential adsorption of lactoglobulin from the protein mixture. In many systems, gamma serw at high protein concentrations even becomes negative due to the strong competition of BSA and water for binding to the surface sites in the presence of lactoglobulin. For lactoglobulin-gelatin mixtures, adsorption of both proteins is enhanced as protein concentration is increased until limiting values for adsorption are reached. Beyond the limiting value, lactoglobulin is further accumulated at the interface without limit when protein concentration is high. For gelatin-albumin mixtures, extent of gelatin adsorption increases with increase in the adsorption of BSA. The limit for saturation of adsorption for gelatin is not reached for many systems. At acid pH, adsorbed BSA appears to be desorbed from the surface in the presence of gelatin. From the results thus obtained the role of electrostatic and hydrophobic effects in controlling the adsorption process has been analysed.     

Polarity as a criterion in protein design

Hypothetical proteins can be tested computationally by determining whether or not the designed sequence-structure pair has the characteristics of a typical globular protein. We have developed such a test by deriving quantities with approximately constant value for all globular proteins, based on empirical analysis of the exposed and buried surfaces of 128 structurally known proteins. The characteristic quantities that best appear to segregate badly designed or deliberately misfolded proteins from their properly folded natural relatives are the polar fraction of side chains on the protein surface and, independently, in the protein interior. Three of the seven hypothetical structures tested here can be rejected as having too many polar side-chain groups in the interior or too few on the protein surface. In addition, a recently designed nutritional protein is identified as being very much unlike globular proteins. These database-derived characteristic quantities are useful in screening designed proteins prior to experiment and may be useful in screening experimentally determined (X-ray, NMR) protein structures for possible errors.     

A systematic approach to the comparison of protein structures

A systematic method has been developed for comparing the backbone conformations of proteins (Remington & Matthews, 1978). Two proteins are compared by successively optimizing the agreement between all possible segments of a chosen length from one protein, and all possible segments of the same length from the other protein. The method reveals any similarities between the two proteins, and provides an estimate of the statistical significance of any given structure agreement that is obtained.The method has been tested in a number of cases, including comparisons of the dehydrogenases and of the pancreatic and bacterial serine proteases. These examples were chosen to test the ability of the comparison method to detect structural similarities in the presence of large insertions and deletions. The results suggest that the detection of the “nucleotide binding fold” in the dehydrogenases is at the limit of the capability of the comparison technique in its original form, although it may be possible to generalize the method to allow for insertions and deletions in proteins.The results of many protein comparisons, made with different probe lengths, are summarized. For medium and long probe lengths, the average value of the structural agreement does not depend very much on the type of protein being compared. The average value of the structure agreement increases with the square root of the probe length, but for probe lengths above about 40 residues, the standard deviation is independent of probe length. From these observations it is possible to construct a generalized probability diagram to evaluate the significance of any structure agreement that might be obtained in comparing two proteins.     

Identification of a novel hepatic calcium binding protein

Calcium binding activity in the 100,000 X g supernatant of bovine liver has been isolated by a procedure involving DEAE cellulose and Sephadex G-100 chromatography. In addition to calmodulin, two new high affinity calcium binding proteins have been identified. On gel filtration chromatography these proteins migrate with apparent molecular weights of 83,700 and 51,400; whereas by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the two proteins migrate identically with Mr 63,000. In the presence of millimolar Mg2+, both proteins bind up to one mol Ca2+/mol protein. Half-maximal binding occurs at approximately 0.1 microM Ca2+. Amino acid compositional analysis reveals that both proteins are acidic, and contain about 40% glx and asx. Peptide mapping procedures suggest that these proteins may be highly homologous or multiple forms of a single protein. The results show the existence of calcium binding protein(s) other than calmodulin in hepatic cytosol.     

Reconstruction of 3D structures from protein contact maps

The prediction of the protein tertiary structure from solely its residue sequence (the so called Protein Folding Problem) is one of the most challenging problems in Structural Bioinformatics. We focus on the protein residue contact map. When this map is assigned it is possible to reconstruct the 3D structure of the protein backbone. The general problem of recovering a set of 3D coordinates consistent with some given contact map is known as a unit-disk-graph realization problem and it has been recently proven to be NP-Hard. In this paper we describe a heuristic method (COMAR) that is able to reconstruct with an unprecedented rate (3-15 seconds) a 3D model that exactly matches the target contact map of a protein. Working with a non-redundant set of 1760 proteins, we find that the scoring efficiency of finding a 3D model very close to the protein native structure depends on the threshold value adopted to compute the protein residue contact map. Contact maps whose threshold values range from 10 to 18 Ångstroms allow reconstructing 3D models that are very similar to the proteins native structure.     

Inter-residue interactions in protein folding and stability

During the process of protein folding, the amino acid residues along the polypeptide chain interact with each other in a cooperative manner to form the stable native structure. The knowledge about inter-residue interactions in protein structures is very helpful to understand the mechanism of protein folding and stability. In this review, we introduce the classification of inter-residue interactions into short, medium and long range based on a simple geometric approach. The features of these interactions in different structural classes of globular and membrane proteins, and in various folds have been delineated. The development of contact potentials and the application of inter-residue contacts for predicting the structural class and secondary structures of globular proteins, solvent accessibility, fold recognition and ab initio tertiary structure prediction have been evaluated. Further, the relationship between inter-residue contacts and protein-folding rates has been highlighted. Moreover, the importance of inter-residue interactions in protein-folding kinetics and for understanding the stability of proteins has been discussed. In essence, the information gained from the studies on inter-residue interactions provides valuable insights for understanding protein folding and de novo protein design.     

High quality protein microarray using in situ protein purification

Background  

In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.     

A successful change of circumstance: a transition state for membrane protein folding

Knowledge of the transition state is key to understanding a reaction mechanism. This vital information has been lacking for integral membrane protein folding, but now recent advances have given insight into the structure of their folding transition state. This progress has arisen through the successful translation of a classical protein engineering method, ? value analysis, from water-soluble proteins to the hydrophobic, membrane-embedded protein class. This review covers the transition state for the folding of α helical membrane proteins. Helix formation in the transition state correlates with sequence position and the order of transmembrane insertion into the cell membrane, showing that in vitro measurements, in entirely different conditions to natural membranes, may reflect the cellular situation.     

Side chain dynamics and alternative hydrogen bonding in the mechanism of protein thermostabilization

To elucidate the mechanism of protein thermostabilization, the thermodynamic properties of small monomeric proteins from mesophilic and thermophilic organisms have been analyzed. Molecular dynamics simulations were employed in the study of dynamic features of charged and polar side chains of amino acid residues. The basic conclusion has been made: surface charged and polar side chains with high conformational mobility can form alternative hydrogen bonded (H-bonded) donor-acceptor pairs. The correlation between the quantitative content of alternative H-bonds per residue and the temperature of maximal thermostability of proteins has been found. The proposed mechanism of protein thermostabilization suggests continuous disruption of the primary H-bonds and formation of alternative ones, which maintain constant the enthalpy value in the native state and prevent a rapid increase of the conformational entropy with the rising temperature. The analysis of the results show that the more residues located in the N- and C-terminal regions and in the extended loops that are capable of forming alternative longer-range H-bonded pairs, the higher the protein thermostability.