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). / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / November 12, 2014. / cryo-electron microscopy, electron tomography, matlab, myosin, smooth muscle, structural biology / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Dissertation; P. Bryant Chase, Committee Member; Thomas C. S. Keller, Committee Member; Hong Li, Committee
Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_252878 |
| Contributors | Qin, Zhuan (authoraut), 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) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource (191 pages), computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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