Andrew Coulson

Sequence homologies between hsp60 and autoantigens

The human heat shock protein (hsp) 60 shares sequence homology with a wide range of autoantigens including those of insulin dependent diabetes mellitus, Hashimotos thyroiditis, glomerulonephritis, scleroderma, pemphigoid, rheumatoid arthritis, multiple sclerosis, chronic active hepatitis, primary biliary cirrhosis and Addisons disease. Here we show the extent of this homology and suggest that it contributes to autoimmunity through cross-reactivity between hsp60 and tissue-specific proteins containing similar epitope motifs. Differences between individuals in MHC class II may influence the selection of a particular hsp60 epitope and the corresponding target antigen that gives rise to an autoimmune disease.

Why Is Carbonic Anhydrase Essential to Escherichia coli

The can (previously yadF) gene of Escherichia coli encodes a beta-class carbonic anhydrase (CA), an enzyme which interconverts CO(2) and bicarbonate. Various essential metabolic processes require either CO(2) or bicarbonate and, although carbon dioxide and bicarbonate spontaneously equilibrate in solution, the low concentration of CO(2) in air and its rapid diffusion from the cell mean that insufficient bicarbonate is spontaneously made in vivo to meet metabolic and biosynthetic needs. We calculate that demand for bicarbonate is 10(3)- to 10(4)-fold greater than would be provided by uncatalyzed intracellular hydration and that enzymatic conversion of CO(2) to bicarbonate is therefore necessary for growth. We find that can expression is ordinarily required for growth in air. It is dispensable if the atmospheric partial pressure of CO(2) is high or during anaerobic growth in a closed vessel at low pH, where copious CO(2) is generated endogenously. CynT, the single E. coli Can paralog, can, when induced with azide, replace Can; also, the gamma-CA from Methanosarcina thermophila can at least partially replace it. Expression studies showed that can transcription does not appear to respond to carbon dioxide concentration or to be autoregulated. However, can expression is influenced by growth rate and the growth cycle; it is expressed best in slow-growing cultures and at higher culture densities. Expression can vary over a 10-fold range during the growth cycle and is also elevated during starvation or heat stress.

Predicted disulfide-bonded structures for three uniquely related proteins of Plasmodium falciparum, Pfs230, Pfs4845 and Pf12

Pfs230 is a surface protein of the gametes of Plasmodium falciparum and has been demonstrated to be a target of malaria transmission-blocking antibodies; it is an important candidate antigen for a transmission-blocking vaccine. The target epitopes of transmission-blocking antibodies against Pfs230 are almost all reduction sensitive suggesting that disulfide bonds are critical for folding the native molecule. Following the cloning of the Pfs230 gene attempts are now underway to express subunits of the protein for use in vaccine trials. It will be important to understand the disulfide-bond structure of the Pfs230 to achieve this goal. In this paper we present a model for this structure based on the observation that the Pfs230 molecule contains a series of regularly repeated cysteine-containing motifs. Four such motifs have been identified, together with a fifth cysteineless motif, which occur in the same relative order, with regular alternating omission of specific motifs, 14 times throughout the length of the protein. Each of the 14 sets of motifs contains an even number of cysteine residues (2, 4 or 6). We postulate that each set folds into a separate disulfide-bonded domain in which corresponding pairs of cysteines form an equivalent disulfide bond in every such domain. The postulated bonding arrangements in the different domains are mutually confirmatory throughout the sequence of Pfs230. We have identified two other malaria proteins, Pfs48/45 and Pf12, which share the same arrangements of motifs and conform to the same disulfide-bond structure proposed for Pfs230; no other proteins in the sequence data base share these characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

The lytB Gene of Escherichia coli Is Essential and Specifies a Product Needed for Isoprenoid Biosynthesis

LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.

Significance of protein sequence similarities.

Publisher Summary The aim of database searching is to pick out the cases of significant similarity from a larger background of unrelated sequences. By definition, therefore, most of the sequences in a database are not significantly related to the query sequence. The method depends only on collecting enough results to have an adequate representation of the ‘by chance’ distribution. The parameters of the distribution depend on the frequency distribution of residues in the query sequence. If this is non-uniform within the sequence, then the improbability of a given match will also vary within the sequence. Three types of search algorithms have been analyzed statistically. The simplest is the comparison of every subsequence of a fixed “window” length from the query sequence with every such subsequence from one or more reference sequences. Each window comparison is scored by summing the appropriate score for each pair of aligned amino acid residues. The significance of the maximum observed similarity can then be assessed in terms of the expected values.

A proposed structure for ‘Family 18’ chitinases a possible function for narbonin

The sequence of narbonin, a leguminous seed protein with the TIM barrel structure but of unknown function, is significantly similar to endo‐β‐N‐acetylglucosaminidase H from Streptomyces plicatus. This protein is a member of a family of chitinases, ‘Family 18’ of the glycosyl hydrolases. It is proposed that the catalytic domain of this family has the TIM barrel structure. It is proposed that narbonin has chitinase activity, or has been derived from a chitinase by loss of function.

Segments of bacteriophage λ (orf221) and φ80 are homologous to genes coding for mammalian protein phosphatases

Abstract The amino acid sequences of mammalian protein phosphatase 1 and 2A were compared pairwise with every sequence in the National Biomedical Research Foundation protein sequence database using an exhaustive searching programme [Coulson et al., Comp. J. 30 (1987) 420–424], The N-terminal half of the protein encoded by an open reading frame, orf 221, in bacteriophage λ (nt 43224–43886 in the map of Daniels et al. [in Hendrix et al. (Eds.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 519–676] shows 35% identity to either protein phosphatase 1 or 2A in this region. If conservative replacements are included the overall homology rises to 49%. A gene in φ80 also shows 35% identity with the mammalian protein phosphatases. The results indicate that orf 221 of phage λ and the homologous φ80 gene may encode protein phosphatases. The possible roles of protein phosphorylation in the propagation of bacteriophage are discussed.

Protein folding in Escherichia coli: the chaperonin GroE and its substrates

A brief summary of the role of DnaK and GroE chaperones in protein folding precedes a discussion of the role of GroE in Escherichia coli. We consider its obligate substrates, the 8 that are both obligate and essential, and the prospects for constructing a mutant that could survive without it. Structural features of GroE-dependent polypeptides are also considered.

Evolving Protein Similarity Scoring Matrices Using Differential Evolution

The problem of protein structure prediction remains one of the major unsolved problems in structural biochemistry. The most successful method to date for predicting protein tertiary (3-dimensional) structure from the primary sequence data (of amino acids) is homology modelling based on alignment with similar sequences of known structure. The premier component utilised in this process is a scoring matrix that determines how similar one protein is to another.