Does the S2 Rod of Myosin II Uncoil upon Two-Headed Binding to Actin? A Leucine-Zippered HMM Study

Tania Chakrabarty, Chris Yengo, Corry Baldacchino, Li Qiong Chen, H. Lee Sweeney, Paul R. Selvin

Research output: Contribution to journalArticle

Abstract

Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tendd to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.

Original languageEnglish (US)
Pages (from-to)12886-12892
Number of pages7
JournalBiochemistry
Volume42
Issue number44
DOIs
StatePublished - Nov 11 2003

Fingerprint

Myosin Type II
Myosin Subfragments
Leucine
Actins
Myosins
Head
Smooth Muscle Myosins
Leucine Zippers
Actin Cytoskeleton
Dimers
Adenosine Diphosphate
Freezing
Melting point
Quenching
Nucleotides
Fluorescence
Amino Acids
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Does the S2 Rod of Myosin II Uncoil upon Two-Headed Binding to Actin? A Leucine-Zippered HMM Study. / Chakrabarty, Tania; Yengo, Chris; Baldacchino, Corry; Chen, Li Qiong; Sweeney, H. Lee; Selvin, Paul R.

In: Biochemistry, Vol. 42, No. 44, 11.11.2003, p. 12886-12892.

Research output: Contribution to journalArticle

Chakrabarty, T, Yengo, C, Baldacchino, C, Chen, LQ, Sweeney, HL & Selvin, PR 2003, 'Does the S2 Rod of Myosin II Uncoil upon Two-Headed Binding to Actin? A Leucine-Zippered HMM Study', Biochemistry, vol. 42, no. 44, pp. 12886-12892. https://doi.org/10.1021/bi035144f
Chakrabarty, Tania ; Yengo, Chris ; Baldacchino, Corry ; Chen, Li Qiong ; Sweeney, H. Lee ; Selvin, Paul R. / Does the S2 Rod of Myosin II Uncoil upon Two-Headed Binding to Actin? A Leucine-Zippered HMM Study. In: Biochemistry. 2003 ; Vol. 42, No. 44. pp. 12886-12892.
@article{8e4ce1708d2b462ba98e4081f4e93660,
title = "Does the S2 Rod of Myosin II Uncoil upon Two-Headed Binding to Actin? A Leucine-Zippered HMM Study",
abstract = "Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of {"}zippered{"} dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tendd to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated {"}on{"} state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.",
author = "Tania Chakrabarty and Chris Yengo and Corry Baldacchino and Chen, {Li Qiong} and Sweeney, {H. Lee} and Selvin, {Paul R.}",
year = "2003",
month = "11",
day = "11",
doi = "10.1021/bi035144f",
language = "English (US)",
volume = "42",
pages = "12886--12892",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "44",

}

TY - JOUR

T1 - Does the S2 Rod of Myosin II Uncoil upon Two-Headed Binding to Actin? A Leucine-Zippered HMM Study

AU - Chakrabarty, Tania

AU - Yengo, Chris

AU - Baldacchino, Corry

AU - Chen, Li Qiong

AU - Sweeney, H. Lee

AU - Selvin, Paul R.

PY - 2003/11/11

Y1 - 2003/11/11

N2 - Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tendd to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.

AB - Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tendd to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.

UR - http://www.scopus.com/inward/record.url?scp=0242507464&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0242507464&partnerID=8YFLogxK

U2 - 10.1021/bi035144f

DO - 10.1021/bi035144f

M3 - Article

C2 - 14596602

AN - SCOPUS:0242507464

VL - 42

SP - 12886

EP - 12892

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 44

ER -