Dataset Information

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551.

ABSTRACT: In the cytochrome c-551 family, the heme 17-propionate caboxylate group is always hydrogen bonded to an invariant Trp-56 and conserved residues (His and Arg mainly, Lys occasionally) at position 47. The mutation of His-47 to Ala-47 for Pseudomas stutzeri ZoBell cytochrome c-551 removes this otherwise invariant hydrogen bond. The solution structure of ferrous H47A has been solved based on NMR-derived constraints. Results indicate that the mutant has very similar main chain folding compared to wild-type. However, less efficient packing of residues in the mutant surrounding the heme propionates leads to more solvent exposure for both propionate groups, which may account for decreased stability of the mutant. The mutant has a reduction potential different from wild-type, and furthermore, the pH dependence of this potential is not the same as for wild-type. The structure of the mutant suggests that these changes are related to the loss of the residue-47 propionate hydrogen bond and the loss of charge on the side chain of residue 47.

SUBMITTER: Liang Q

PROVIDER: S-EPMC1948052 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551.

Liang Qiaoli Q Miller Gregory T GT Beeghley Chanda A CA Graf Coyner B CB Timkovich Russell R

Biophysical journal 20070511 5

In the cytochrome c-551 family, the heme 17-propionate caboxylate group is always hydrogen bonded to an invariant Trp-56 and conserved residues (His and Arg mainly, Lys occasionally) at position 47. The mutation of His-47 to Ala-47 for Pseudomas stutzeri ZoBell cytochrome c-551 removes this otherwise invariant hydrogen bond. The solution structure of ferrous H47A has been solved based on NMR-derived constraints. Results indicate that the mutant has very similar main chain folding compared to wil ...[more]

PMID: 17496029

Similar Datasets

Project description:UnlabelledMetal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu(2+) and Zn(2+) resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu(2+) and Zn(2+) resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu(2+) and Zn(2+) resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu(2+) resistance systems do not show decreased fitness relative to the wild type when exposed to Cu(2+) In addition, new genes are identified that have no known connection to Zn(2+) (corB, corC, Psest_3226, Psest_3322, and Psest_0618) or Cu(2+) resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB, corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu(2+)- and Zn(2+)-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu(2+) and Zn(2+) efflux and resistance and their effects on denitrifying metabolism.ImportanceIn this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca(2+)/H(+) antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg(2+) transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB, corC, or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.

Project description:BackgroundStudies on membrane proteins are often hampered by insufficient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins.ResultsHere we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosa. Pseudomonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several effective antibiotics were identified and a protocol for plasmid transformation was established. We present a workflow including cloning of the target proteins, small-scale screening for the best production conditions and finally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purification of a Gluconate:H+ Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated.ConclusionsPseudomonas stutzeri is an alternative production host for membrane proteins with success rates comparable to E. coli. However, some proteins were produced with high yields in P. stutzeri but not in E. coli and vice versa. Therefore, P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins. Using the new pL2020 vector no additional cloning is required to test both hosts in parallel.

Dataset Information

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551.

Publications

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets