Determination of the primary structure of homologous proteins by sequence analysis of peptide mixtures

We propose a rapid method to determine the primary structure of a protein knowing the sequence of a homologous protein. The method consists in submitting both the reduced and alkylated proteins to an enzymatic or chemical hydrolysis and performing the sequence analysis of the peptide mixtures. The assessment of the unknown sequence and the degree of identity of the two proteins are reached by comparing the two sequence analyses. The sequences of all the possible peptides present in the two mixtures are reconstructed and the differences in the two sequences are determined. If necessary, the differences can be confirmed by performing a mass spectrometric analysis of the two mixtures. We used this procedure on two homologous proteins of known sequence to furnish an application example of the method.     

Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures.

We set up a method to get rapid information on the three-dimensional structure of peptide and proteins of known sequence. Both native and alkylated polypeptide is hydrolyzed with a number of proteases at different digestion times and the resulting mixtures are compared by HPLC analysis to establish the differences in the hydrolysis pathways of the folded and unfolded molecule. Then, the unfractionated digestion mixtures of the native polypeptide are submitted to automatic sequence analysis to identify the hydrolysis sites. The sequence of each fragment present in the mixtures is reconstructed and its amount determined by quantitative data of the sequence analyses. We used this approach to determine the amino acid surface accessibility of wheatwin1, a pathogenesis-related protein from wheat, and constructed a predictive three-dimensional model based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. The procedure allowed us to quickly identify and quantify the hydrolysis at the susceptible bonds which could be classified as exposed, partially hidden, or inaccessible. The results were useful to evidentiate and discuss concordances and differences between experimental and model predicted accessibilities of amino acid residues. Proteins 1999;36:192-204.     

NdPASA: a pairwise sequence alignment server for distantly related proteins

SUMMARY: NdPASA is a web server specifically designed to optimize sequence alignment between distantly related proteins. The program integrates structure information of the template sequence into a global alignment algorithm by employing neighbor-dependent propensities of amino acids as a unique parameter for alignment. NdPASA optimizes alignment by evaluating the likelihood of a residue pair in the query sequence matching against a corresponding residue pair adopting a particular secondary structure in the template sequence. NdPASA is most effective in aligning homologous proteins sharing low percentage of sequence identity. The server is designed to aid homologous protein structure modeling. A PSI-BLAST search engine was implemented to help users identify template candidates that are most appropriate for modeling the query sequences.     

Protein identification in complex mixtures

This paper investigates the prospects of successful mass spectrometric protein identification based on mass data from proteolytic digests of complex protein mixtures. Sets of proteolytic peptide masses representing various numbers of digested proteins in a mixture were generated in silico. In each set, different proteins were selected from a protein sequence collection and for each protein the sequence coverage was randomly selected within a particular regime (15-30% or 30-60%). We demonstrate that the Probity algorithm, which is characterized by an optimal tolerance for random interference, employed in an iterative procedure can correctly identify >95% of proteins at a desired significance level in mixtures composed of hundreds of yeast proteins under realistic mass spectrometric experimental constraints. By using a model of the distribution of protein abundance, we demonstrate that the very high efficiency of identification of protein mixtures that can be achieved by appropriate choices of informatics procedures is hampered by limitations of the mass spectrometric dynamic range. The results stress the desire to choose carefully experimental protocols for comprehensive proteome analysis, focusing on truly critical issues such as the dynamic range, which potentially limits the possibilities of identifying low abundance proteins.     

Protein identification from product ion spectra of peptides validated by correlation between measured and predicted elution times in liquid chromatography/mass spectrometry

Reversed-phase liquid chromatography (LC) directly coupled with electrospray-tandem mass spectrometry (MS/MS) is a successful choice to obtain a large number of product ion spectra from a complex peptide mixture. We describe a search validation program, ScoreRidge, developed for analysis of LC-MS/MS data. The program validates peptide assignments to product ion spectra resulting from usual probability-based searches against primary structure databases. The validation is based only on correlation between the measured LC elution time of each peptide and the deduced elution time from the amino acid sequence assigned to product ion spectra obtained from the MS/MS analysis of the peptide. Sufficient numbers of probable assignments gave a highly correlative curve. Any peptide assignments within a certain tolerance from the correlation curve were accepted for the following arrangement step to list identified proteins. Using this data validation program, host protein candidates responsible for interaction with human hepatitis B virus core protein were identified from a partially purified protein mixture. The present simple and practical program complements protein identification from usual product ion search algorithms and reduces manual interpretation of the search result data. It will lead to more explicit protein identification from complex peptide mixtures such as whole proteome digests from tissue samples.     

A differential geometric treatment of protein structure comparison

The technique of model-building a protein of known sequence but unknown tertiary structure from the structures of homologous proteins is probably so far the most reliable means of mapping from primary to tertiary structure. A key step towards the realization of the aim is to develop ways of aligning three-dimensional structures of homologus proteins, thereby deriving the rules useful for protein modelling. We have developed a generalized differential-geometric representation of protein local conformation for use in a protein comparison program which aligns protein sequences on the basis of their sequence and conformational knowledge. Because the differetial-geometric distance measure between local conformations is independent of the coordinate frame and remains chirality information, the comparison program is easily implemented, relatively rational and reasonably fast. The utility of this program for aligning closely and distantly related homologous proteins is demonstrated by multiple alignment of globins, serine proteinases and aspartic proteinase domains. Particularly, the method has reached the rational alignment between the mammalian and microbial serine proteinases as compared with many published alignment programs.     

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)¹ is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment².Coagulation Factor VIII (FVIII)³ is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting⁴. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete⁵. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa^6,7.In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.     

PALI-a database of Phylogeny and ALIgnment of homologous protein structures

PALI (release 1.2) contains three-dimensional (3-D) structure-dependent sequence alignments as well as structure-based phylogenetic trees of homologous protein domains in various families. The data set of homologous protein structures has been derived by consulting the SCOP database (release 1.50) and the data set comprises 604 families of homologous proteins involving 2739 protein domain structures with each family made up of at least two members. Each member in a family has been structurally aligned with every other member in the same family (pairwise alignment) and all the members in the family are also aligned using simultaneous super-position (multiple alignment). The structural alignments are performed largely automatically, with manual interventions especially in the cases of distantly related proteins, using the program STAMP (version 4.2). Every family is also associated with two dendrograms, calculated using PHYLIP (version 3.5), one based on a structural dissimilarity metric defined for every pairwise alignment and the other based on similarity of topologically equivalent residues. These dendrograms enable easy comparison of sequence and structure-based relationships among the members in a family. Structure-based alignments with the details of structural and sequence similarities, superposed coordinate sets and dendrograms can be accessed conveniently using a web interface. The database can be queried for protein pairs with sequence or structural similarities falling within a specified range. Thus PALI forms a useful resource to help in analysing the relationship between sequence and structure variation at a given level of sequence similarity. PALI also contains over 653 'orphans' (single member families). Using the web interface involving PSI_BLAST and PHYLIP it is possible to associate the sequence of a new protein with one of the families in PALI and generate a phylogenetic tree combining the query sequence and proteins of known 3-D structure. The database with the web interfaced search and dendrogram generation tools can be accessed at http://pauling.mbu.iisc.ernet. in/ approximately pali.     

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.     

Gene-protein relationships in the flagellar hook-basal body complex of Bacillus subtilis: sequences of the flgB, flgC, flgG, fliE and fliF genes

The nucleotide sequence of five genes from the major Bacillus subtilis chemotaxis locus has been determined. Four of these genes encode proteins that are homologous to the Salmonella typhimurium FlgB, FlgC, FlgG and FliF proteins. One gene encodes a protein that is homologous to the Escherichia coli FliE protein. The data from S. typhimurium and E. coli suggest that all of these proteins form part of the hook-basal body (HBB) complex of the bacterial flagella. The FlgB, FlgC and FlgG proteins are components of the proximal and distal rods. The FliF protein forms the M-ring that anchors the rod assembly to the membrane. The role of the FliE protein within the HBB complex has not yet been determined. The similarity between the B. subtilis and S. typhimurium proteins suggests that the structure of the M-ring and the rod may be similar in the two species. However, we observed some differences in size and amino acid composition between some of the corresponding homologues that suggest the basal body proteins may be organized slightly differently within B. subtilis.     

Homology modeling of 3D structure of human chitinase-like protein CHI3L2

The human genome encodes six proteins of family 18 glycosyl hydrolases, two active chitinases and four chitinase-like lectins (chi-lectins) lacking catalytic activity. The present article is dedicated to homology modeling of 3D structure of human chitinase 3-like 2 protein (CHI3L2), which is overexpressed in glial brain tumors, and its structural comparison with homologous chi-lectin CHI3L1. Two crystal structures of CHI3L1 in free state (Protein Data Bank codes 1HJX and 1NWR) were used as structural templates for the homology modeling by Modeller 9.7 program, and the best quality model structure was selected from the obtained model ensemble. Analysis of potential oligosaccharide-binding groove structures of CHI3L1 and CHI3L2 revealed significant differences between these two homologous proteins. 8 of 19 amino acid residues important for ligand binding are substituted in CHI3L2: Tyr³⁴/Asp³⁹, Trp⁶⁹/Lys⁷⁴, Trp⁷¹/Lys⁷⁶, Trp⁹⁹/Tyr¹⁰⁴, Asn¹⁰⁰/Leu¹⁰⁵, Met²⁰⁴/Leu²¹⁰, Tyr²⁰⁶/Phe²¹² and Arg²⁶³/His²⁷¹. The differences between these residues could influence the structure of the ligand-binding groove and substantially change the ability of CHI3L2 to bind oligosaccharide ligands.     

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.     

CHORAL: a differential geometry approach to the prediction of the cores of protein structures

MOTIVATION: Although the cores of homologous proteins are relatively well conserved, amino acid substitutions lead to significant differences in the structures of divergent superfamilies. Thus, the classification of amino acid sequence patterns and the selection of appropriate fragments of the protein cores of homologues of known structure are important for accurate comparative modelling. RESULTS: CHORAL utilizes a knowledge-based method comprising an amalgam of differential geometry and pattern recognition algorithms to identify conserved structural patterns in homologous protein families. Propensity tables are used to classify and to select patterns that most likely represent the structure of the core for a target protein. In our benchmark, CHORAL demonstrates a performance equivalent to that of MODELLER.     

Examining protein surface structure in highly conserved sequence variants with mass spectrometry

A simple mass spectrometry-based method capable of examining protein structure called SNAPP (selective noncovalent adduct protein probing) is used to evaluate the structural consequences of point mutations in naturally occurring sequence variants from different species. SNAPP monitors changes in the attachment of noncovalent adducts to proteins as a function of structural state. Mutations that lead to perturbations to the electrostatic surface structure of a protein affect noncovalent attachment and are easily observed with SNAPP. Mutations that do not alter the tertiary structure or electrostatic surface structure yield similar results by SNAPP. For example, bovine, porcine, and human insulin all have very similar backbone structures and no basic or acidic residue mutations, and the SNAPP distributions for all three proteins are very similar. In contrast, four variants of cytochrome c (cytc) have varying degrees of sequence homology, which are reflected in the observed SNAPP distributions. Bovine and pigeon cytc have several basic or acidic residue substitutions relative to horse cytc, but the SNAPP distributions for all three proteins are similar. This suggests that these mutations do not significantly influence the protein surface structure. On the other hand, yeast cytc has the least sequence homology and exhibits a unique, though related, SNAPP distribution. Even greater differences are observed for lysozyme. Hen and human lysozyme have identical tertiary structures but significant variations in the locations of numerous basic and acidic residues. The SNAPP distributions are quite distinct for the two forms of lysozyme, suggesting significant differences in the surface structures. In summary, SNAPP experiments are relatively easy to perform, require minimal sample consumption, and provide a facile route for comparison of protein surface structure between highly homologous proteins.     

Identifying sequence determinants of reduction potentials of metalloproteins

The reduction potential of an electron transfer protein is one of its most important functional characteristics. Although the type of redox site and the protein fold are the major determinants of the reduction potential of a redox-active protein, its amino acid sequence may tune the reduction potential as well. Thus, homologous proteins can often be divided into different classes, with each class characterized by a biological function and a reduction potential. Site-specific mutagenesis of the sequence determinants of the differences in the reduction potential between classes should change the reduction potential of a protein in one class to that of the other class. Here, a procedure is presented that combines energetic and bioinformatic analysis of homologous proteins to identify sequence determinants that are also good candidates for site-specific mutations, using the [4Fe–4S] ferredoxins and the [4Fe–4S] high-potential iron–sulfur proteins as examples. This procedure is designed to guide site-specific mutations or more computationally expensive studies, such as molecular dynamics simulations. To make the procedure more accessible to the general scientific community, it is being implemented into CHARMMing, a Web-based portal, with a library of density functional theory results for the redox site that are used in the setting up of Poisson–Boltzmann continuum electrostatics calculations for the protein energetics.     

To Investigate Protein Evolution by Detecting Suppressed Epitope Structures

Material remains of ancestor nucleotides and proteins are largely unavailable, thus sequence comparison among homologous genes in present-day organisms forms the core of current knowledge of molecular evolution. Variation in protein three-dimensional structure is a basis for functional diversity. To study the evolution of three-dimensional structures in related proteins would significantly improve our understanding of protein evolution and function. A protein may contain ancestor conformations that have been allosterically suppressed by evolutionarily additive structures. Using monoclonal antibody probes to detect such conformation in proteins after removing the suppressor structure, our study demonstrates three-dimensional structure evidence for the evolutionary relationship between troponin I and troponin T, two subunits of the troponin complex in the Ca²⁺-regulatory system of striated muscle, and among their muscle type-specific isoforms. The experimental data show the feasibility of detecting evolutionarily suppressed history-telling structural states in proteins by removing conformational modulator segments added during evolution. In addition to identifying structural modifications that were critical to the emergence of diverged proteins, investigating this novel mode of evolution will help us to understand the origin and functional potential of protein structures.     

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.     

Definition and characterization of a "trypsinosome" from specific peptide characteristics by nano-HPLC-MS/MS and in silico analysis of complex protein mixtures

Although HPLC-ESI-MS/MS is rapidly becoming an indispensable tool for the analysis of peptides in complex mixtures, the sequence coverage it affords is often quite poor. Low protein expression resulting in peptide signal intensities that fall below the limit of detection of the MS system in combination with differences in peptide ionization efficiency plays a significant role in this. A second important factor stems from differences in physicochemical properties of each peptide and how these properties relate to chromatographic retention and ultimate detection. To identify and understand those properties, we compared data from experimentally identified peptides with data from peptides predicted by in silico digest of all corresponding proteins in the experimental set. Three different complex protein mixtures extracted were used to define a training set to evaluate the amino acid retention coefficients based on linear regression analysis. The retention coefficients were also compared with other previous hydrophobic and retention scale. From this, we have constructed an empirical model that can be readily used to predict peptides that are likely to be observed on our HPLC-ESI-MS/MS system based on their physicochemical properties. Finally, we demonstrated that in silico prediction of peptides and their retention coefficients can be used to generate an inclusion list for a targeted mass spectrometric identification of low abundance proteins in complex protein samples. This approach is based on experimentally derived data to calibrate the method and therefore may theoretically be applied to any HPLC-MS/MS system on which data are being generated.     

Identification of local conformational similarity in structurally variable regions of homologous proteins using protein blocks

Structure comparison tools can be used to align related protein structures to identify structurally conserved and variable regions and to infer functional and evolutionary relationships. While the conserved regions often superimpose well, the variable regions appear non superimposable. Differences in homologous protein structures are thought to be due to evolutionary plasticity to accommodate diverged sequences during evolution. One of the kinds of differences between 3-D structures of homologous proteins is rigid body displacement. A glaring example is not well superimposed equivalent regions of homologous proteins corresponding to α-helical conformation with different spatial orientations. In a rigid body superimposition, these regions would appear variable although they may contain local similarity. Also, due to high spatial deviation in the variable region, one-to-one correspondence at the residue level cannot be determined accurately. Another kind of difference is conformational variability and the most common example is topologically equivalent loops of two homologues but with different conformations. In the current study, we present a refined view of the "structurally variable" regions which may contain local similarity obscured in global alignment of homologous protein structures. As structural alphabet is able to describe local structures of proteins precisely through Protein Blocks approach, conformational similarity has been identified in a substantial number of 'variable' regions in a large data set of protein structural alignments; optimal residue-residue equivalences could be achieved on the basis of Protein Blocks which led to improved local alignments. Also, through an example, we have demonstrated how the additional information on local backbone structures through protein blocks can aid in comparative modeling of a loop region. In addition, understanding on sequence-structure relationships can be enhanced through our approach. This has been illustrated through examples where the equivalent regions in homologous protein structures share sequence similarity to varied extent but do not preserve local structure.     

ORB, a homology-based program for the prediction of protein NMR chemical shifts

A computer program (ORB) has been developed to predict ¹H,¹³C and ¹⁵N NMR chemical shifts of previouslyunassigned proteins. The program makes use of the information contained in achemical shift database of previously assigned proteins supplemented by astatistically derived averaged chemical shift database in which the shifts arecategorized according to their residue, atom and secondary structure type[Wishart et al. (1991) J. Mol. Biol., 222, 311–333]. The predictionprocess starts with a multiple alignment of all previously assigned proteinswith the unassigned query protein. ORB uses the sequence and secondarystructure alignment program XALIGN for this task [Wishart et al. (1994)CABIOS, 10, 121–132; 687–688]. The prediction algorithm in ORB isbased on a scoring of the known shifts for each sequence. The scores dependon global sequence similarity, local sequence similarity, structuralsimilarity and residue similarity and determine how much weight one particularshift is given in the prediction process. In situations where no applicablepreviously assigned chemical shifts are available, the shifts derived from theaveraged database are used. In addition to supplying the user with predictedchemical shifts, ORB calculates a confidence value for every prediction. Theseconfidence values enable the user to judge which predictions are the mostaccurate and they are particularly useful when ORB is incorporated into acomplete autoassignment package. The usefulness of ORB was tested on threemedium-sized proteins: an interleukin-8 analog, a troponin C synthetic peptideheterodimer and cardiac troponin C. Excellent results are obtained if ORB isable to use the chemical shifts of at least one highly homologous sequence.ORB performs well as long as the sequence identity between proteins with knownchemical shifts and the new sequence is not less than 30%.