Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Autoren

  • Doreen E Culham
  • Yaroslava Vernikovska
  • Natalia Tschowri
  • Robert A B Keates
  • Janet M Wood
  • Joan M Boggs

Organisationseinheiten

Externe Organisationen

  • University of Guelph
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Details

OriginalspracheEnglisch
Seiten (von - bis)13584-93
Seitenumfang10
FachzeitschriftBiochemistry
Jahrgang47
Ausgabenummer51
PublikationsstatusVeröffentlicht - 23 Dez. 2008

Abstract

ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.

Zitieren

Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality. / Culham, Doreen E; Vernikovska, Yaroslava; Tschowri, Natalia et al.
in: Biochemistry, Jahrgang 47, Nr. 51, 23.12.2008, S. 13584-93.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Culham, D. E., Vernikovska, Y., Tschowri, N., Keates, R. A. B., Wood, J. M., & Boggs, J. M. (2008). Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality. Biochemistry, 47(51), 13584-93. https://doi.org/10.1021/bi801576x
Culham DE, Vernikovska Y, Tschowri N, Keates RAB, Wood JM, Boggs JM. Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality. Biochemistry. 2008 Dez 23;47(51):13584-93. doi: 10.1021/bi801576x
Culham, Doreen E ; Vernikovska, Yaroslava ; Tschowri, Natalia et al. / Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality. in: Biochemistry. 2008 ; Jahrgang 47, Nr. 51. S. 13584-93.
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@article{e0cf33b11c6f4a41a1f0e917ad756b11,
title = "Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality",
abstract = "ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.",
keywords = "Animals, Biological Transport, Cattle, Escherichia coli/metabolism, Escherichia coli Proteins/chemistry, Membrane Transport Proteins/chemistry, Models, Biological, Molecular Conformation, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Protons, Symporters/chemistry, Water/metabolism",
author = "Culham, {Doreen E} and Yaroslava Vernikovska and Natalia Tschowri and Keates, {Robert A B} and Wood, {Janet M} and Boggs, {Joan M}",
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language = "English",
volume = "47",
pages = "13584--93",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "51",

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TY - JOUR

T1 - Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality

AU - Culham, Doreen E

AU - Vernikovska, Yaroslava

AU - Tschowri, Natalia

AU - Keates, Robert A B

AU - Wood, Janet M

AU - Boggs, Joan M

PY - 2008/12/23

Y1 - 2008/12/23

N2 - ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.

AB - ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.

KW - Animals

KW - Biological Transport

KW - Cattle

KW - Escherichia coli/metabolism

KW - Escherichia coli Proteins/chemistry

KW - Membrane Transport Proteins/chemistry

KW - Models, Biological

KW - Molecular Conformation

KW - Protein Conformation

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Protons

KW - Symporters/chemistry

KW - Water/metabolism

U2 - 10.1021/bi801576x

DO - 10.1021/bi801576x

M3 - Article

C2 - 19049385

VL - 47

SP - 13584

EP - 13593

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 51

ER -

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