On ’The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils’ by James D. Potter

Abstract

After the discovery of troponin by Ebashi almost sixty years ago the field of striated muscle regulation has made significant progress. In the 1970’s the nascent troponin field gained momentum, including contributions by James D. Potter who established the stoichiometry of contractile proteins in the myofibril (Arch Biochem Biophys. 1974 Jun; 162(2):436-41. https://doi.org/10.1016/0003-9861(7490202-1)). This opened the door to refinement of competing models that described possible thick filament configurations. This study suggested the presence of one myosin per cross bridge and provided accurate calculations of the molar ratios of each protein - myosin: actin: tropomyosin: troponin T: troponin I: troponin C.

The troponin field was established in 1966 when Ebashi et al. described a protein complex in striated muscle that governs actomyosin calcium sensitivity [1] and found that it consists of two components troponin (Tn) and tropomyosin (Tm) [2,3]. A few years later in 1970 Potter published a study in Archives of Biochemistry and Biophysics that showed that Tn and Tm were present and quantifiable in embryonic chick leg muscle [4]. In 1974 Potter described the precise relationship between troponin subunits and the myofibril.

Dr. James D. Potter is a distinguished scientist in the field of muscle regulation and former Professor and Chairman of the University of Miami, Miller School of Medicine. In a recent interview with Dr. Potter on March 15, 2022, we asked him to describe the scientific backdrop of his 1974 Archives in Biochemistry and Biophysics (ABB) article that is being commemorated in the ABB 80^th Anniversary Special Issue. When asked about this publication Potter said, “it’s a relatively simple little paper, but some important numbers came out of it.” He mentioned that one of the greatest challenges in the field at that time was developing a method to accurately determine the molar ratios and stoichiometries of myofibrillar proteins. In brief, Potter’s 1974 ABB paper established the stoichiometry and approximate molar ratios of myofibril proteins as 1:7:1:1:1:1 for myosin, actin, Tm, and Tn subunits C, I, and T respectively. At the time, some controversy existed regarding the correct stoichiometry of these proteins. In a study published in 1973 by Ebashi et al. it was assumed that the molar ratios of the Tn subunits were proportional while Sperling et al., in 1979 had obtained a molar ratio for TnT:TnI:TnC of 1:2:1 for isolated troponin [5]. In 1983 Yates et al. obtained results in agreement with Potter (1974) and Ebashi (1973) by comparing molecular weights obtained from amino acid sequence and reported average molar ratios for actin:TnT:TnI:TnC of 6.99:1.27:1.08:0.99 [6].

In the featured 1974 Archives in Biochemistry and Biophysics article, Potter reported a key discovery that not all myofibrillar proteins bound the same to Fast Green. According to others, under alkaline conditions Fast Green binds via electrostatic interactions to basic amino acid side chains of strongly basic proteins. Therefore, it provides a valid quantification method of basic but not acidic proteins [7,8]. For this reason, the Fast Green protein quantification method underreported the amount of acidic proteins such as TnC, which binds half as much dye as Tm. To address this issue Potter generated highly purified standards for these myofibril proteins using the Kjeldahl method of protein total nitrogen analysis. This yielded an accurate method for calculating molar ratios of myofibrillar proteins when utilizing photometric methods such as Fast Green. When describing the methodologies utilized in this study Potter explained several crucial factors for the accurate assessment of myofibrillar protein content including starting with clean, quickly prepared high-quality myofibril preparations. Tn subunits are particularly sensitive to proteolytic degradation, a complicating factor for determining correct stoichiometry [5,9]. In addition, the myofibril preparations were assessed for ATPase function prior to use to assure they possessed the full range of Ca²⁺ sensitivity. The amount of myofibril proteins run on gels was reduced to avoid saturation of actin bands so that accurate densiometric quantifications could be obtained. Around the same time, Potter published another study that helped define known positions assumed by Tm on actin thin filaments during contraction and relaxation [10]. From these studies he proposed a model that explained how Ca²⁺ and the Tn complex regulates muscle contraction. In addition, in 1975 Potter elucidated the role that the Tn complex Ca²⁺ and Mg²⁺ binding sites played -in myofibrillar ATPase activity [11].

In summary, Potter’s 1974 Archives in Biochemistry and Biophysics article defined the stoichiometry of myofibril proteins, a finding that has stood the test of time, however, it also answered additional questions. For example, until this study it was unclear whether there were one or two myosin molecules per cross bridge and the number of myosin molecules per thick filament was under debate [12-14]. This paper provided convincing evidence that “there would not be more than one myosin molecule per crossbridge.” This newly defined stoichiometric ratio allowed Potter to calculate the number of myosins per thick filament – and estimate 254 myosin molecules per thick filament [15]. This was close to Squire’s three-stranded model, which proposed 264 single myosin cross bridges [12]. While the featured paper helped solve myofibrillar protein stoichiometry and the number of myosin molecules per crossbridge, the question of whether the thick filament helical structure was two-stranded or three-stranded persisted. Therefore, the stoichiometric information provided by this article was valuable for refining models of vertebrate skeletal muscle thick filaments. In summary, researchers in the striated muscle field are beneficiaries of Dr. Potter’s visionary contributions to the muscle regulation field in the 1970s that provided fundamental insights into the mechanisms governing striated muscle regulation.