title: A Novel Method of Determining the Functional Effects of a Minor Genetic Modification of a Protein.
abstract: Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being "kicked" by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either "Standard" (Std) (decaying monotonically in time) or "Non-standard" (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.
authors.affiliation: Department of Cell Biology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center , Fort Worth, TX , USA.
authors.forename: Janhavi
authors.initials: J
authors.lastname: Nagwekar
authors.affiliation: Department of Cell Biology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center , Fort Worth, TX , USA.
authors.forename: Divya
authors.initials: D
authors.lastname: Duggal
authors.affiliation: Department of Cell Biology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center , Fort Worth, TX , USA.
authors.forename: Krishna
authors.initials: K
authors.lastname: Midde
authors.affiliation: Department of Mathematics, Computer Science, and Physics, Texas Wesleyan University , Fort Worth, TX , USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Rich
authors.affiliation: Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami , Miami, FL , USA.
authors.forename: Jingsheng
authors.initials: J
authors.lastname: Liang
authors.affiliation: Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami , Miami, FL , USA.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kazmierczak
authors.affiliation: Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami , Miami, FL , USA.
authors.forename: Wenrui
authors.initials: W
authors.lastname: Huang
authors.affiliation: Department of Cell Biology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center , Fort Worth, TX , USA.
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
authors.affiliation: Department of Cell Biology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center , Fort Worth, TX , USA.
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
authors.affiliation: Department of Physics and Astronomy, Texas Christian University , Fort Worth, TX , USA.
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.affiliation: Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami , Miami, FL , USA.
authors.forename: Danuta
authors.initials: D
authors.lastname: Szczesna-Cordary
authors.affiliation: Department of Cell Biology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center , Fort Worth, TX , USA.
authors.forename: Julian
authors.initials: J
authors.lastname: Borejdo
keywords: actin; autocorrelation function; fluorescence polarization; low expressing mutations; myosin light chain; phalloidin-actin