Prediction of the location of structural domains in globular proteins

The location of structural domains in proteins is predicted from the amino acid sequence, based on the analysis of a computed contact map for the protein, the average distance map (ADM). Interactions between residues i and j in a protein are subdivided into several ranges, according to the separation |i-j| in the amino acid sequence. Within each range, average spatial distances between every pair of amino acid residues are computed from a data base of known protein structures. Infrequently occurring pairs are omitted as being statistically insignificant. The average distances are used to construct a predicted ADM. The ADM is analyzed for the occurrence of regions with high densities of contacts (compact regions). Locations of rapid changes of density between various parts of the map are determined by the use of scanning plots of contact densities. These locations serve to pinpoint the distribution of compact regions. This distribution, in turn, is used to predict boundaries of domains in the protein. The technique provides an objective method for the location of domains both on a contact map derived from a known three-dimensional protein structure, the real distance map (RDM), and on an ADM. While most other published methods for the identification of domains locate them in the known three-dimensional structure of a protein, the technique presented here also permits the prediction of domains in proteins of unknown spatial structure, as the construction of the ADM for a given protein requires knowledge of only its amino acid sequence.     

In vitro and in silico cloning of Xenopus laevis SOD2 cDNA and its phylogenetic analysis

By using the methodology of both wet and dry biology (i.e., RT-PCR and cycle sequencing, and biocomputational technology, respectively) and the data obtained through the Genome Projects, we have cloned Xenopus laevis SOD2 (MnSOD) cDNA and determined its nucleotide sequence. These data and the deduced protein primary structure were compared with all the other SOD2 nucleotide and amino acid sequences from eukaryotes and prokaryotes, published in public databases. The analysis was performed by using both Clustal W, a well known and widely used program for sequence analysis, and AntiClustAl, a new algorithm recently created and implemented by our group. Our results demonstrate a very high conservation of the enzyme amino acid sequence during evolution, which proves a close structure-function relationship. This is to be expected for very ancient molecules endowed with critical biological functions, performed through a specific structural organization. The nucleotide sequence conservation is less pronounced: this too was foreseeable, due to neutral mutations and to the species-specific codon usage. The data obtained by using AntiClustAl are comparable with those produced with Clustal W, which validates this algorithm as an important new tool for biocomputational analysis. Finally, it is noteworthy that evolutionary trees, drawn by using all the available data on SOD2 nucleotide sequences and amino acid and either Clustal W or AntiClustAl, are comparable to those obtained through phylogenetic analysis based on fossil records.     

Characterization of a pollen-specific cDNA clone from Zea mays and its expression.

A pollen-specific cDNA clone, Zmc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

Conformational preference functions for predicting helices in membrane proteins

A suite of FORTRAN programs, PREF, is described for calculating preference functions from the data base of known protein structures and for comparing smoothed profiles of sequence-dependent preferences in proteins of unknown structure. Amino acid preferences for a secondary structure are considered as functions of a sequence environment. Sequence environment of amino acid residue in a protein is defined as an average over some physical, chemical, or statistical property of its primary structure neighbors. The frequency distribution of sequence environments in the data base of soluble protein structures is approximately normal for each amino acid type of known secondary conformation. An analytical expression for the dependence of preferences on sequence environment is obtained after each frequency distribution is replaced by corresponding Gaussian function. The preference for the α-helical conformation increases for each amino acid type with the increase of sequence environment of buried solvent-accessible surface areas. We show that a set of preference functions based on buried surface area is useful for predicting folding motifs in α-class proteins and in integral membrane proteins. The prediction accuracy for helical residues is 79% for 5 integral membrane proteins and 74% for 11 α-class soluble proteins. Most residues found in transmembrane segments of membrane proteins with known α-helical structure are predicted to be indeed in the helical conformation because of very high middle helix preferences. Both extramembrane and transmembrane helices in the photosynthetic reaction center M and L subunits are correctly predicted. We point out in the discussion that our method of conformational preference functions can identify what physical properties of the amino acids are important in the formation of particular secondary structure elements. © 1993 John Wiley & Sons, Inc.     

Transporters for amino acids in plant cells: some functions and many unknowns

Membrane proteins are essential to move amino acids in or out of plant cells as well as between organelles. While many putative amino acid transporters have been identified, function in nitrogen movement in plants has only been shown for a few proteins. Those studies demonstrate that import systems are fundamental in partitioning of amino acids at cellular and whole plant level. Physiological data further suggest that amino acid transporters are key-regulators in plant metabolism and that their activities affect growth and development. By contrast, knowledge on the molecular mechanisms of cellular export processes as well as on intracellular transport of amino acids is scarce. Similarly, little is known about the regulation of amino acid transporter function and involvement of the transporters in amino acid signaling. Future studies need to identify the missing components to elucidate the importance of amino acid transport processes for whole plant physiology and productivity.     

Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study

Understanding the molecular basis for the enhanced stability of proteins from thermophiles has been hindered by a lack of structural data for homologous pairs of proteins from thermophiles and mesophiles. To overcome this difficulty, complete genome sequences from 9 thermophilic and 21 mesophilic bacterial genomes were aligned with protein sequences with known structures from the protein data bank. Sequences with high homology to proteins with known structures were chosen for further analysis. High quality models of these chosen sequences were obtained using homology modeling. The current study is based on a data set of models of 900 mesophilic and 300 thermophilic protein single chains and also includes 178 templates of known structure. Structural comparisons of models of homologous proteins allowed several factors responsible for enhanced thermostability to be identified. Several statistically significant, specific amino acid substitutions that occur going from mesophiles to thermophiles are identified. Most of these are at solvent-exposed sites. Salt bridges occur significantly more often in thermophiles. The additional salt bridges in thermophiles are almost exclusively in solvent-exposed regions, and 35% are in the same element of secondary structure. Helices in thermophiles are stabilized by intrahelical salt bridges and by an increase in negative charge at the N-terminus. There is an approximate decrease of 1% in the overall loop content and a corresponding increase in helical content in thermophiles. Previously overlooked cation-pi interactions, estimated to be twice as strong as ion-pairs, are significantly enriched in thermophiles. At buried sites, statistically significant hydrophobic amino acid substitutions are typically consistent with decreased side chain conformational entropy.     

The relationship of DNA base composition and individual protein composition in micro-organisms

Summary To investigate the dependence of protein composition on DNA base composition, a set of data on individual proteins with known amino acid compositions from a spectrum of bacterial species has been compiled. It is found that similar relationships of amino acid frequency to G + C content exist for these proteins as for the bulk proteins studied by Sueoka (1961). The data are analysed by linear and cubic regression, and a measure of the proportions of A + T-rich and G + C-rich codons in the underlying messenger RNAs is put forward. The theoretical limits on the G + C content of coding DNA are discussed, and inference are made about the various selective forces acting on DNAs of different G + C contents.     

Complete amino acid sequence of a new murine T-cell growth factor P40

A new murine T-cell growth factor, designated P40, which supports growth of helper T-cells in the absence of interleukin-2, interleukin-4 and antigen has been isolated from helper T-cell lines in sufficient quantities (100 micrograms) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed-phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N-terminal sequence data on P40 were unsuccessful due to N-terminal blockage of the native molecule. The nature of this N-terminal blocking was established using a combination of amino acid analysis, fast-atom-bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T-cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict four N-linked glycosylation sites in the P40 molecule.     

Computer modeling studies of the structure of a repressor

Due to advances in molecular biology the DNA sequences of structural genes coding for proteins are often known before a protein is characterized or even isolated. The function of a protein whose amino acid sequence has been deduced from a DNA sequence may not even be known. This has created greater interest in the development of methods to predict the tertiary structures of proteins. The a priori prediction of a protein's structure from its amino acid sequence is not yet possible. However, since proteins with similar amino acid sequences are observed to have similar three-dimensional structures, it is possible to use an analogy with a protein of known structure to draw some conclusions about the structure and properties of an uncharacterized protein. The process of predicting the tertiary structure of a protein relies very much upon computer modeling and analysis of the structure. The prediction of the structure of the bacteriophage 434 cro repressor is used as an example illustrating current procedures.     

The Myofibrillar Protein,Projectin, is Highly Conserved Across Insect Evolution Except for Its PEVK Domain

All striated muscles respond to stretch by a delayed increase in tension. This physiological response, known as stretch activation, is, however, predominantly found in vertebrate cardiac muscle and insect asynchronous flight muscles. Stretch activation relies on an elastic third filament system composed of giant proteins known as titin in vertebrates or kettin and projectin in insects. The projectin insect protein functions jointly as a “scaffold and ruler” system during myofibril assembly and as an elastic protein during stretch activation. An evolutionary analysis of the projectin molecule could potentially provide insight into how distinct protein regions may have evolved in response to different evolutionary constraints. We mined candidate genes in representative insect species from Hemiptera to Diptera, from published and novel genome sequence data, and carried out a detailed molecular and phylogenetic analysis. The general domain organization of projectin is highly conserved, as are the protein sequences of its two repeated regions—the immunoglobulin type C and fibronectin type III domains. The conservation in structure and sequence is consistent with the proposed function of projectin as a scaffold and ruler. In contrast, the amino acid sequences of the elastic PEVK domains are noticeably divergent, although their length and overall unusual amino acid makeup are conserved. These patterns suggest that the PEVK region working as an unstructured domain can still maintain its dynamic, and even its three-dimensional, properties, without the need for strict amino acid conservation. Phylogenetic analysis of the projectin proteins also supports a reclassification of the Hymenoptera in relation to Diptera and Coleoptera. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Non-random relationships among amino acids in protein sequences.

We have computed the observed distribution of amino acid pairs n, n+1 up to n, n+10 within 100 unrelated protein sequences containing a total of 14,034 amino acids. The expected distribution of the same amino acid pairs was calculated assuming that the amino acids within each sequence are arranged in random order. Statistical analysis of the data shows that amino acids in the n, n+1 and n, n+3 relationship deviate from random expectation at the 1% level of significance. These relationships are consistent with known secondary structures; however, there are no amino acid pairs which obviously contribute to the non-random result.     

Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin.

The 'proliferating cell nuclear antigen' (PCNA), also known as cyclin, appears at the G1/S boundary in the cell cycle. Because of its possible relationship with cell proliferation, PCNA/cyclin has been receiving attention. PCNA/cyclin is a non-histone acidic nuclear protein with an apparent mol. wt of 33000-36000. The amino acid composition and the sequence of the first 25 amino acids of rabbit PCNA/cyclin are known. Using an oligonucleotide probe corresponding to the sequence of the first five amino acids, a cDNA clone for PCNA/cyclin was isolated from rat thymocyte cDNA library. The cDNA (1195 bases) contains an open reading frame of 813 nucleotides coding for 261 amino acids. The 3'-non-coding region is 312 nucleotides long and contains three putative polyadenylation signals. The mol. wt of rat PCNA/cyclin was calculated to be 28 748. The deduced amino acid sequence and composition of rat PCNA/cyclin are in excellent agreement with the published data. Using the cDNA probe, two species of mRNA (1.1 and 0.98 kb) were detected in rat thymocyte RNA. Southern blot analysis of total human genomic DNA suggests that there is a single gene coding for PCNA/cyclin. The deduced amino acid sequence of rat PCNA/cyclin has a similarity with that of herpes simplex virus type-1 DNA binding protein.     

Amino Acid Changes in Disease-Associated Variants Differ Radically from Variants Observed in the 1000 Genomes Project Dataset

The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.     

The partial amino acid sequence of the extracellular beta-lactamase I of Bacillus cereus 569/H.

The chemical structure of the extracellular beta-lactamase I of Bacillus cereus 569/H was investigated. Three electrophoretically homogenous charge variants of this enzyme were isolated and amino acid analysis of each revealed no significant differences. However, a degree of N-terminal heterogeneity was found by direct end-group modification of the protein and also on alignment of peptides from tryptic and chymotryptic digestion. The N-terminal heterogeneity observed was great enough to explain the production of the beta-lactamase I isoenzymes which are probably produced by postsynthesis modification of a single gene product. Over 80% of the amino acid sequence of beta-lactamase I was determined by the detailed analysis of peptides derived from tryptic, chymotryptic and thermolytic digests. Five polypeptide fragments were constructed from these data and aligned by comparison with the known amino acid sequences of the penicillinases produced by Bacillus licheniformis and Staphylococcus aureus (Ambler & Meadway, 1969). About 60% of the proposed sequence was identical with that of B. licheniformis penicillinase, whereas the S. aureus enzyme had only about 40% of its residues in common with beta-lactamase I. These results are discussed with reference to the possible evolutionary relationships existing between known beta-lactamases. Detailed evidence for the amino acid sequence proposed has been deposited as Supplementary Publication SUP 50044 (27 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.