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Investigation into the Effect of the S2 Domain of Smooth Muscle Myosin II on Its Interactions with F-actin

Title: An Investigation into the Effect of the S2 Domain of Smooth Muscle Myosin II on Its Interactions with F-actin.
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Name(s): Qin, Zhuan, author
Taylor, Kenneth A., professor directing dissertation
Liu, Xiuwen, 1966-, university representative
Chase, P. Bryant, committee member
Keller, Thomas C. S., committee member
Li, Hong, committee member
Florida State University, degree granting institution
College of Arts and Sciences, degree granting college
Department of Biological Science, degree granting department
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2014
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (191 pages)
Language(s): English
Abstract/Description: Muscle plays a primary role in movement of the body of multicellular organs. A study of muscle contraction at a molecular level will provide understanding of muscular malfunction, as well as insights into the basic mechanism of bimolecular motors. Muscle contraction involves a complex interaction between multiple proteins with multiple domains. Not all of these interactions are well understood. This study is focused on the role of the S2 segment of muscle myosin. S2 is part of the long alpha-helix coiled-coil rod, and plays a significant role in both muscle contraction and myosin II ATPase regulation. In this study, we use the HMM fragment of smooth muscle myosin II (smHMM) which contains a pair of myosin heads held together by the S2 domain. smHMM with a full length S2 shows the ATPase behavior of a fully regulated smooth muscle myosin (smM) but is soluble rather than filamentous. Biochemical studies have shown that the length of the S2 domain affects actin-activated ATPase regulation in smHMM, an observation that would suggest that torsional rigidity of the alpha-helices that comprise S2 is the physical basis because the two heads, which would be nominally on opposite sides of the coiled-coil alpha-helices, must rotate to a position on one side only. However, interpretation of the biochemistry suggested that the effect of S2 length on regulation was due to a requirement that the S2 coiled-coil be long enough to interact with one of the myosin heads in the inactive complex. Modeling studies investigating this effect concluded that torsional rigidity was the explanation. Torsional motions could also be involved in the binding of both myosin heads to a single actin filament because as with formation of the ATPase inhibited conformation, the two heads must come to the same side of the S2 domain. In this case, there is no suggestion of an interaction between the S2 domain and the actin-attached heads and thus an effect can have a simpler interpretation. On that basis, we investigated whether the length of the S2 domain has an effect on the simultaneous binding of both myosin heads to an actin filament using two recombinant smHMM constructs, one with a full length S2 (wt-HMM), the other with a highly shortened S2 with a length of two heptads of S2 followed by a 32 residue leucine zipper (2hepzip-HMM). We compare the amount of 2-headed binding to actin in these two otherwise identical constructs in the complete absence of nucleotide, known as the rigor state, and in the presence of saturating levels of ADP. The myosin S1 head has its strongest binding affinity to actin in the rigor state; while in the presence of ADP, it has less binding affinity. Thus, if torsional rigidity is the physical basis of the effect, the predicted outcome is more 2-headed attachment to actin with wt-HMM-rigor compared to 2hepzip-HMM-rigor, and a less 2-headed attachment for both constructs when ADP is added. For this study, we directly visualize the smHMM attachment to actin filament by combining cryo-electron tomography with subvolume alignment and classification using multivariate data analysis. Methodological advances in several steps were necessary to achieve this goal: (1) segmentation of the subvolumes from the tomograms, (2) alignment of the subvolumes to a feature held in common among all members, and (3) clustering into groups a heterogeneous collection of subvolumes that vary with respect to several criteria. In this study the common feature is the actin filaments, and the heterogeneous feature is the presence or absence of myosin binding to actin by either one head or two. We use a novel approach that utilizes convolution and least squares fitting to smooth the stochastic error in the subvolume centers and Euler angles to improve the alignment. The subvolume alignment was done in a way that enabled all the bound myosin heads to be localized to a single classification site from which the variability can be assessed in a simplified way. Following identification of the bound myosin heads, the different types of attachment are determined that include 2-headed attachments, 1- headed attachments with one free head, which may or may not have an accessible, unlabeled actin subunit nearby, and 1-headed attachments that have no unbound second head, which may arise from dissociation of the S2 domain (Chapter 3). We find that the when the S2 domain is shortened to a length of 2 heptads plus a leucine zipper that 2-headed binding decreases by a factor of 2 compared to full length S2 constructs. This we interpret as evidence of a torsional effect of the S2 helices. Moreover, the addition of ADP, rather than decreasing the amount of 2-headed binding increased it by ~5% in the case of wt-HMM. The effect of ADP addition is compatible with published accounts of ADP addition to smooth muscle fibers, which showed that tension increased by ~3% and stiffness decreased by ~10% (Chapter 4).
Identifier: FSU_migr_etd-9234 (IID)
Submitted Note: A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Fall Semester, 2014.
Date of Defense: November 12, 2014.
Keywords: cryo-electron microscopy, electron tomography, matlab, myosin, smooth muscle, structural biology
Bibliography Note: Includes bibliographical references.
Advisory Committee: Kenneth A. Taylor, Professor Directing Dissertation; P. Bryant Chase, Committee Member; Thomas C. S. Keller, Committee Member; Hong Li, Committee Member.
Subject(s): Biology
Biophysics
Molecular biology
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_migr_etd-9234
Owner Institution: FSU

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Qin, Z. (2014). An Investigation into the Effect of the S2 Domain of Smooth Muscle Myosin II on Its Interactions with F-actin. Retrieved from http://purl.flvc.org/fsu/fd/FSU_migr_etd-9234