Amino acid sequence determination and three‐dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y

Abstract

The complete primary structure of thioredoxin from Rhodobacter sphaeroides Y has been determined by analysis of peptides after cleavage with cyanogen bromide, chymotrypsin and trypsin. Peptides were separated by HPLC and analyzed by liquid-phase and gas-phase sequencer degradations. The protein consists of 105 residues (Mr = 11 180); its amino acid sequence shows a clear homology to the five known thioredoxins from plant or bacterial sources, with 40-56% residue identity when the proteins are aligned at the active-site disulfide. Not only the active-site regions are conserved, but also residues which belong to the hydrophobic surface suggested to be important for binding of procaryote thioredoxins in redox interactions with other proteins (residues 75 -76; 91 -93 in Escherichia coli). A three-dimensional model of Rb. sphaeroides thioredoxin has been derived from the E. coli crystallographic structure with computer graphics. This model indicates that the overall structures as well as the active sites are closely similar; however, the residue substitutions allow both proteins to adopt different local folding as shown in the hydrophobic core.Thioredoxin, a small ubiquitous protein having two redox active half-cystine residues in an exposed active center with the sequence -Cys-Gly-Pro-Cys-, functions as a protein disulfide reductant in a variety of enzymatic reactions [l]. These include the synthesis of 5-aminolaevulinic acid (the first reaction of the bacteriochlorophyll synthesis pathway) in Rhodobacter sphaeroides [2]. In addition, thioredoxin from Escherichia coli participates in phage T7 infection as an essential subunit of the phage-induced DNA polymerase [3]. Recent evidence shows that E. coli thioredoxin-(SH)2 is also essential for the assembly of the single-stranded filamentous phage fl [4, 51. These last two functions have an absolute requirement for thioredoxin as shown by genetic techniques; however, the active-site redox activity is not essential, whereas the reduced conformation of thioredoxin plays so far unknown roles. Furthermore, the hydrophobic surface area of thioredoxin, thought to interact with proteins [6], also seems essential for the filamentous phage assembly machinery [7] and the function of the gene-5 protein in the phage T7 DNA polymerase enzyme [S]. It is, therefore, essential to know the threedimensional structures of thioredoxins in order to understand the mechanisms of regulation achieved by this protein.Complete amino acid sequences are presently known for thioredoxin from five organisms: E. coli [9] [16]. They are highly similar in spite of large differences in amino acid sequence. Models were built for C. nephridii [6] and Anabaena [17] which showed that these two proteins can also adopt similar folding.In the present study we have determined the amino acid sequence of thioredoxin from Rb. sphaeroides Y, and proposed a three-dimensional model derived from the E. coEi crystallographic structure, as a first step toward the understanding of an important aspect of the regulation of ba...

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

“…The thioredoxin of Rhodobacter sphaeroides Y, which lacks histidine in its composition (23), is yet to be tested. 'The diversity of the molecular surfaces of the selected variants of cytochrome c, myoglobin, and lysozyme notwithstanding, their chromatographic behavior on IDA-Cu(II) columns seems to be determined by the absence of an accessible histidyl side chain, an observation consistent with the first notion.…”

The noncompetitive blocker [3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit

“…Automated Edman degradation was carried out in a Beckman 890 C spinning cup sequenator with 0.1 M quadrol in the presence of polybrene as described [2,18]. The sequenator output was dissolved in 2007o acetonitrile and aliquots used for radioactivity measurements and for identification and quantification of phenylthiohydantoin-amino acids by HPLC [18] on a Microbore column with a solvent system similar to that used with the Applied Biosystems 120A PTH analyzer.…”

“…The sequenator output was dissolved in 2007o acetonitrile and aliquots used for radioactivity measurements and for identification and quantification of phenylthiohydantoin-amino acids by HPLC [18] on a Microbore column with a solvent system similar to that used with the Applied Biosystems 120A PTH analyzer. A few degradations were carried out with an Applied Biosystems 470A sequencer, with on-line PTH-amino acid analysis in the 120A analyzer using the standard programs provided by the supplier.…”

Replacement of Pro109 by His in TlpA, a thioredoxin‐like protein from Bradyrhizobium japonicum, alters its redox properties but not its in vivo functions

“…The two thioredoxin‐like domains of PDI are referred to as (1) and (2). References [1, 4–12] are indicated in brackets behind the sequences. The sequence of DsbA from A. vinelandii was derived from the Swiss Prot data base (accession number L76098).…”