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Exploring GTP Control of Bacterial Respiratory System Specific Chaperone Interaction with its Substrate Protein's Twin‐Arginine Signal Peptide
Author(s) -
Cherak Stephana Julia,
Turner Raymond J
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.810.1
Subject(s) - cooperativity , chemistry , chaperone (clinical) , biophysics , protein folding , translocase , dimer , biochemistry , gtp' , twin arginine translocation pathway , crystallography , signal peptide , peptide sequence , enzyme , biology , chromosomal translocation , medicine , organic chemistry , pathology , gene
Many bacterial respiratory redox enzymes depend on the twin‐arginine translocase (Tat) system for translocation and/or membrane insertion. Tat substrates contain an N‐terminal twin‐arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone ‐ redox enzyme maturation protein (REMP) – to help mediate final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin‐arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase (DmsABC). DmsD adopts monomer, dimer, and unique folding forms that appear under pH control. All alternate DmsD forms retain the ability to bind DmsAL. As well, it is now suggested that DmsD cooperatively binds guanosine‐5′‐triphosphate (GTP), which elicits a dissociative effect on the DmsD to DmsAL interaction. Thus, the primary aim of this study was to analyze DmsD thermal stability when subjected to both GTP association and variance in pH. We have utilized two in vitro protein‐protein interaction techniques of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF). DSF experiments, yielding melting temperatures as a determinant of the associated thermal stability, showed an increase in DmsD stabilization upon DmsAL binding. Between pH values of 5.0 and 6.0, DmsD demonstrated the least amount of DmsAL release. This pH range also corresponds to a transition in DmsD folding forms. When DmsD was first subjected to GTP binding, subsequent variance in pH decreased melting cooperativity by the appearance of a pretransition peak, but did not affect melting temperature. However, if alteration in pH occurred prior to GTP binding, the pretransition peak was attenuated, and an incremental increase in melting temperature was observed. These findings illustrate that a greater affinity for DmsAL promotes an increase in DmsD thermal stability. As well, pH shift results in formation of a more uniform DmsD population, with subsequent GTP binding permitting an increase in thermal stability. These results support the idea that H + and GTP play roles as molecular regulators in the biochemical pathway of DmsAB protein maturation – Tat system targeting mechanism. Support or Funding Information Research was supported by a Canadian Institutes of Health Research operating grant ( MOP‐49422 ) to RJT.