TY - JOUR
T1 - Functional and structural characterization of Hyp730, a highly conserved and dormancy-specific hypothetical membrane protein
AU - Fannin, Stewart
AU - Rangel, Jonathan
AU - Bodurin, Abiodun P.
AU - Yu, Tannon
AU - Mistretta, Brandon
AU - Mali, Sujina
AU - Gunaratne, Preethi
AU - Bark, Steven J.
AU - Ebalunode, Jerry O.
AU - Khan, Arshad
AU - Widger, William R.
AU - Sen, Mehmet
N1 - Funding Information:
The work was supported by the National Research University Fund (NRUF‐R110640) and R03 AI139651, and the McCammon Foundation.
Publisher Copyright:
© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
PY - 2021/1
Y1 - 2021/1
N2 - Membrane proteins represent major drug targets, and the ability to determine their functions, structures, and conformational changes will significantly advance mechanistic approaches to both biotechnology and bioremediation, as well as the fight against pathogenic bacteria. A pertinent example is Mycobacterium tuberculosis (H37Rv), which contains ~4000 protein-coding genes, with almost a thousand having been categorized as ‘membrane protein’, and a few of which (~1%) have been functionally characterized and structurally modeled. However, the functions and structures of most membrane proteins that are sparsely, or only transiently, expressed, but essential in small phenotypic subpopulations or under stress conditions such as persistence or dormancy, remain unknown. Our deep quantitative proteomics profiles revealed that the hypothetical membrane protein 730 (Hyp730) WP_010079730 (protein ID Mlut_RS11895) from M. luteus is upregulated in dormancy despite a ~5-fold reduction in overall protein diversity. Its H37Rv paralog, Rv1234, showed a similar proteomic signature, but the function of Hyp730-like proteins has never been characterized. Here, we present an extensive proteomic and transcriptomic analysis of Hyp730 and have also characterized its in vitro recombinant expression, purification, refolding, and essentiality as well as its tertiary fold. Our biophysical studies, circular dichroism, and tryptophan fluorescence are in immediate agreement with in-depth in silico 3D-structure prediction, suggesting that Hyp730 is a double-pass membrane-spanning protein. Ablation of Hyp730-expression did not alter M. luteus growth, indicating that Hyp730 is not essential. Structural homology comparisons showed that Hyp730 is highly conserved and non-redundant in G+C rich Actinobacteria and might be involved, under stress conditions, in an energy-saving role in respiration during dormancy.
AB - Membrane proteins represent major drug targets, and the ability to determine their functions, structures, and conformational changes will significantly advance mechanistic approaches to both biotechnology and bioremediation, as well as the fight against pathogenic bacteria. A pertinent example is Mycobacterium tuberculosis (H37Rv), which contains ~4000 protein-coding genes, with almost a thousand having been categorized as ‘membrane protein’, and a few of which (~1%) have been functionally characterized and structurally modeled. However, the functions and structures of most membrane proteins that are sparsely, or only transiently, expressed, but essential in small phenotypic subpopulations or under stress conditions such as persistence or dormancy, remain unknown. Our deep quantitative proteomics profiles revealed that the hypothetical membrane protein 730 (Hyp730) WP_010079730 (protein ID Mlut_RS11895) from M. luteus is upregulated in dormancy despite a ~5-fold reduction in overall protein diversity. Its H37Rv paralog, Rv1234, showed a similar proteomic signature, but the function of Hyp730-like proteins has never been characterized. Here, we present an extensive proteomic and transcriptomic analysis of Hyp730 and have also characterized its in vitro recombinant expression, purification, refolding, and essentiality as well as its tertiary fold. Our biophysical studies, circular dichroism, and tryptophan fluorescence are in immediate agreement with in-depth in silico 3D-structure prediction, suggesting that Hyp730 is a double-pass membrane-spanning protein. Ablation of Hyp730-expression did not alter M. luteus growth, indicating that Hyp730 is not essential. Structural homology comparisons showed that Hyp730 is highly conserved and non-redundant in G+C rich Actinobacteria and might be involved, under stress conditions, in an energy-saving role in respiration during dormancy.
KW - Micrococcus luteus
KW - bacteria
KW - dormancy
KW - transmembrane-spanning helices
KW - Amino Acid Sequence
KW - Porins/genetics
KW - Gene Expression Regulation, Bacterial/genetics
KW - Gene Expression Profiling
KW - RNA, Messenger/genetics
KW - Tandem Mass Spectrometry
KW - Bacterial Proteins/metabolism
KW - Mycobacterium tuberculosis/genetics
KW - Genome, Bacterial/genetics
KW - Membrane Proteins/metabolism
KW - Latent Infection/genetics
KW - Proteomics/methods
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U2 - 10.1002/mbo3.1154
DO - 10.1002/mbo3.1154
M3 - Article
C2 - 33650800
AN - SCOPUS:85101984021
SN - 2045-8827
VL - 10
SP - e1154
JO - MicrobiologyOpen
JF - MicrobiologyOpen
IS - 1
M1 - e1154
ER -