Thermostabilization of protein A-luciferase fusion protein by single amino acid mutation

A gene expression plasmid, pMALU7, for coding a fusion protein between protein A (SpA) and mutated firefly luciferase (Luc), was constructed. The fused gene was expressed in Escherichia coli and the resulting protein (SpA-LucTS) was purified with affinity chromatography. By changing a single amino acid, from Glu to Lys at the position 354 in the luciferase moiety, the thermostability of luciferase was improved.     

Short inverse complementary amino acid sequences generate protein complexity

Inversions of short genomic sequences play a central role in the generation of protein complexity. More than half of the 1300 motifs registered in ProSite have protein inverse complementary sequences (princoms) among proteins registered in SwissProt. The observed number of princoms occurrences exceeds by far the expected number (p < 10(-10)). Princoms often endow their host proteins with a whole new range of biochemical and physiological capabilities, including the possibility of intramolecular and intermolecular disulfide bond formation. These results support the idea that, like the duplications, the inversions of small genomic fragments have been a fundamental mechanism for shaping genomes.     

Integrated displays of aligned amino acid sequences and protein structures

Programs for the Sun-4 workstation permit the combined displayof a table of aligned amino acid sequences of a family of proteins,and a corresponding three-dimensional fold. Interactive facilitiesinclude the ability, to scroll through the sequences, to rotatethe structure and to connect the examination of the sequencesand the structure by selecting a portion of the sequences andautomatically highlighting the corresponding region in the structureand vice versa. These programs are well suited to support applications suchas the investigation of the structural or functional significanceof conserved patterns of amino acids in the sequences of a familyof proteins. Received on July 3, 1990; accepted on January 25, 1991     

Phylogeny reconstruction for protein sequences based on amino acid properties

This paper presents an essentially new method used to construct phylogenetic trees from related amino acid sequences. The method is based on a new distance measure which describes sequence relationships by means of typical steric and physicochemical properties of the amino acids and is advantageous in some essential points. The method was applied to different sets of protein sequences and the results were compared with other well-established methods.     

A simple intact protein analysis by MALDI-MS for characterization of ribosomal proteins of two genome-sequenced lactic acid bacteria and verification of their amino acid sequences

Rapid identification of bacteria by a bioinformatics-based approach, which processes the mass spectra observed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), relies on the calculated masses of ribosomal subunit proteins as biomarkers predicted from amino acid sequences found in protein sequence databases. To verify the actual state of the registered sequence information, a simple intact protein analysis by MALDI-MS using cell lysates as samples was applied to the characterization of ribosomal proteins from genome-sequenced Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains. This method avoided the risk of loss of some subunit proteins and the formation of disulfide bonds during the purification of ribosomal proteins. By comparing this with the MALDI mass spectra of different strains and carrying out manual inspection of sequence information, a total of five errors in N-terminal amino acid sequences were identified. After sequence correction, approximately 40 out of 53 subunit proteins could be assigned, considering N-terminal methionine loss only as a post-translational modification. These show promise for use as practical biomarkers for the rapid identification of S. thermophilus and L. bulgaricus. After verification of these amino acid sequences, mass differences relative to those of genome-sequenced strains have the potential for distinguishing bacteria at the strain level.     

A sensitive procedure to compare amino acid sequences

Methods are discussed that provide sensitive criteria for detection of weak sequence homologies. They are based on the Dayhoff relatedness odds amino acid exchange matrix and certain residue physical characteristics. The search procedure uses several residue probe lengths in comparing all possible segments of two protein sequences, and search plots are shown with peak values displayed over the entire search length. Alignments are automatically effected using the highest search matrix values and without the necessity of gap penalties. Tests for significance are derived from actual protein sequences rather than a random shuffling procedure.     

Similar amino acid sequences revisited

The rapid accumulation of protein sequences, many bearing unexpected resemblances to each other, is providing a new perspective on evolution.     

Identification of latent periodicity in amino acid sequences of protein families

For detection of the latent periodicity of the protein families responsible for various biological functions, methods of information decomposition, cyclic profile alignment, and the method of noise decomposition have been used. The latent periodicity, being specific to a particular family, is recognized in 94 of 110 analyzed protein families. Family specific periodicity was found for more than 70% of amino acid sequences in each of these families. Based on such sequences the characteristic profile of the latent periodicity has been deduced for each family. Possible relationship between the recognized latent periodicity, evolution of proteins, and their structural organization is discussed.     

An algorithm to classify amino acid sequences into protein groups of Bothrops jararacussu venomous gland

An algorithm for automatic clustering of database protein sequences from Bothrops jararacussu venomous gland, according to sequence similarities of their domains, is described. The program was written in C and Perl languages. This algorithm compares a domain with each ORF protein sequence in the database. Each nucleotide FASTA sequence generates six ORFs. As a result, the user has a list containing all sequences found in a specific domain and a display of the sequence, domain and number of hits. The algorithm lists only the sequences that present a minimum similarity of 30 hits and the best alignment. This limit was considered appropriate. The algorithm is available in the Internet (www.compbionet.org.br/cgi-domains/homesnake) and it can quickly and accurately organizes large database into classes.     

Enhancement of essential amino acid contents in crops by genetic engineering and protein design

The importance and urgency of providing humans and animals with quality proteins are reflected in the growing scientific and industrial interest in augmenting the nutritive value of the world's protein sources. Such nutritive value is determined by the protein content in 'essential amino acids', those that cannot be synthesized de novo and that must be supplied from the diet. It is the object of this review to discuss recent advances in the genetic modification of crops that aim to provide enhanced quantities of essential amino acids.     

Statistical potential-based amino acid similarity matrices for aligning distantly related protein sequences

Aligning distantly related protein sequences is a long-standing problem in bioinformatics, and a key for successful protein structure prediction. Its importance is increasing recently in the context of structural genomics projects because more and more experimentally solved structures are available as templates for protein structure modeling. Toward this end, recent structure prediction methods employ profile-profile alignments, and various ways of aligning two profiles have been developed. More fundamentally, a better amino acid similarity matrix can improve a profile itself; thereby resulting in more accurate profile-profile alignments. Here we have developed novel amino acid similarity matrices from knowledge-based amino acid contact potentials. Contact potentials are used because the contact propensity to the other amino acids would be one of the most conserved features of each position of a protein structure. The derived amino acid similarity matrices are tested on benchmark alignments at three different levels, namely, the family, the superfamily, and the fold level. Compared to BLOSUM45 and the other existing matrices, the contact potential-based matrices perform comparably in the family level alignments, but clearly outperform in the fold level alignments. The contact potential-based matrices perform even better when suboptimal alignments are considered. Comparing the matrices themselves with each other revealed that the contact potential-based matrices are very different from BLOSUM45 and the other matrices, indicating that they are located in a different basin in the amino acid similarity matrix space.     

Predicting amino acid residues responsible for enzyme specificity solely from protein sequences

We describe a general, modular method for developing protocols to identify the amino acid residues that most likely define the division of a protein superfamily into two subsets. As one possibility, we use PROBE to gather superfamily members and perform an ungapped alignment. We then use a modified BLOSUM62 substitution matrix to determine the discriminating power of each column of aligned residues. The overall method is particularly useful for predicting amino acids responsible for substrate or binding specificity when no structures are available. We apply our method to three pairs of protein classes in three different superfamilies, and present our results, some of which have been experimentally verified. This approach may accelerate the elucidation of enzymic substrate specificity, which is critical for both mechanistic insights into biocatalysis and ultimate application.     

DIVAA: analysis of amino acid diversity in multiple aligned protein sequences

MOTIVATION: Multiple alignments of proteins are an effective way of identifying conserved amino acids that provide clues to functional relationships among proteins. Quantitation of the abundances of amino acids found at each position in a sequence motif can provide a basis for understanding the structural and functional constraints at each point. Distribution of information across a motif has been used previously, but the non-intuitive nature of the analysis has limited its impact. RESULTS: Here, we introduce a quantitative measure of amino acid sequence diversity (DIVAA) that has a simple, intuitive meaning. Diversity, as a measure of sequence conservation or variation, is inextricably linked to the probability of selecting identical pairs from a distribution. We demonstrate its utility through the analysis of four populations: ATP-binding P-loops, hypervariable domains of kappa light chains, signal sequences, and the N- and C- termini of proteins. DIVAA provides a simple means to generate hypotheses concerning the contribution of individual residues to the functional and evolutionary relationships among proteins. AVAILABILITY: Access to DIVAA software is available at RELIC (http://relic.bio.anl.gov).     

A single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion

The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F(1) portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.