Hylary R. Trayer

Size and charge requirements for kinetic modulation and actin binding by alkali 1-type myosin essential light chains.

The alkali 1-type isoforms of myosin essential light chains from vertebrate striated muscles have an additional 40 or so amino acids at their N terminus compared with the alkali 2-type. Consequently two light chain isoenzymes of myosin subfragment-1 can be isolated. Using synthesized peptide mimics of the N-terminal region of alkali 1-type essential light chains, we have found by1H NMR that the major actin binding region occurred in the N-terminal four residues, APKK. . . . . These results were confirmed by mutating this region of the human atrial essential light chain, resulting in altered actin-activated MgATPase kinetics when the recombinant light chains were hybridized into rabbit skeletal subfragment 1. Substitution of either Lys3 or Lys4 with Ala resulted in increased K m and k cat and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys4 with Asp reduced actin binding and increased K m andk cat still further. Alteration of Ala1 to Val did not alter the kinetic parameters of the hybrid subfragment 1 or the essential light chain’s ability to bind actin. Furthermore, we found a significant correlation between the apparent K m for actin and thek cat for MgATP turnover for each mutant hybrid, strengthening our belief that the binding of actin by alkali 1-type essential light chains results directly in modulation of the myosin motor.

Role of the myosin light chains in binding to actin

Myosin from the fast twitch muscles of rabbit is hexameric and comprises two polypeptide chains of approx. mol. wt 200 000 (heavy chains) and 4 mol (per mol myosin) of light chains: two identical phosphorylatable polypeptides of mol. wt 18 000 (the P-LC) and two polypeptides of molecular weight 22 000 and 16 000 (the so-called alkali light chains, Al and A2 respectively (see review [l] and references therein). The four light chain components reside in the globular ‘heads’ of the myosin molecule, the subfragment 1 (S-l) regions, which possess both the actin binding and ATPase activities of the myosin [ 1 ] . Densitometric and radiochemical methods have shown that there is an unequal distribution of the two alkali light chains which supports the hypothesis that myosin isoenzymes exist [2,3]. Myosin from chicken breast-muscle also contains three species of light chain and it is likely that this myosin is similar to that of the rabbit in overall structure and design. Chymotryptic digestion of insoluble myosin filaments from rabbit fast-twitch muscle produces S-l species without the P-LC and such fragments have been separated into two species by ion-exchange chromatography, each of which contains a single type of alkali light chain [4]. The precise role of the myosin light chains is still

Differential binding of rabbit fast muscle myosin light chain isoenzymes to regulated actin

The direct binding of S1(A1) and S1(A2) to regulated actin has been investigated by centrifugation. Binding was measured in the presence of either Mg·AdoPP[NH]P or Mg·ADP at 24°C at various ionic strengths. At low ionic strength, in either the presence or absence of Ca2+, the binding of S1(A1) to regulated actin was always stronger than for S1(A2). As the ionic strength was increased the differential binding between S1(A1) and S1(A2) was still maintained in the presence of Ca2+ but not in its absence. These data are discussed in terms of a modifying role for the N‐terminal region of the A1 light chain in regulation of the contractile process.

The Importance of the Carboxyl-terminal Domain of Cardiac Troponin C in Ca2+-sensitive Muscle Regulation

The interactions between troponin I and troponin C are central to the Ca2+-regulated control of striated muscle. Using isothermal titration microcalorimetry we have studied the binding of human cardiac troponin C (cTnC) and its isolated domains to human cardiac troponin I (cTnI). We provide the first binding data for these proteins while they are free in solution and unmodified by reporter groups. Our data reveal that the C-terminal domain of cTnC is responsible for most of the free energy change upon cTnC·cTnI binding. Importantly, the interaction between cTnI and the C-terminal domain of cTnC is 8-fold stronger in the presence of Ca2+ than in the presence of Mg2+, suggesting that the C-terminal domain of cTnC may play a modulatory role in cardiac muscle regulation. Changes in the affinity of cTnI for cTnC and its isolated C-terminal domain in response to ionic strength support this finding, with both following similar trends. At physiological ionic strength the affinity of cTnC for cTnI changed very little in response to Ca2+, although the thermodynamic data show a clear distinction between binding in the presence of Ca2+and in the presence of Mg2+.

A new and rapid method for the isolation of myosin from small amounts of muscle and non-muscle tissue by affinity chromatography.

The immobilization of adenosine nucleotide derivatives [l] has proved to be a general affinity chromatographic medium which can be used for a variety of enzymes. Insolubilized AMP derivatives have been used extensively both to purify and study the mode of action of dehydrogenase [l-4] whereas agarose conjugates of ADP and ATP derivatives have proved excellent media for the chromatography of kinases [ 1,5] and ATPases [ 1,6-81. The contractile protein, myosin, with its requirement to interact reversibly with both actin and adenosine nucleotides during muscle contraction, represents an ideal case in which to exploit this technique. Conventional methods for purifying this protein are based upon the fact that large quantities of starting muscle tissue are usually available and they are not easily adaptable to the situation where either the supply of muscle is limiting or myosin is to be prepared from other sources. In this report we describe a method of purifying myosin direct from tissue extracts by affinity chromatography on immobilized ADP derivatives which can be readily applied to isolating this protein from very small quantities of muscle tissue or from non-muscle sources.

Calcium and peptide binding to folded and unfolded conformations of cardiac Troponin C. Electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry

The binding of Ca2+ and of a peptide (N-TnC) to human cardiac troponin C (TnC) and its isolated N- and C-terminal domains has been characterized by electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry. The peptide N-TnI corresponds to residues 1–29 of the cardiac-specific N-terminal extension of human cardiac troponin I (TnI). The binding of Ca2+ to intact TnC in the absence of the peptide was found to take a bimodal form with preferred stoichiometries of 1:1 TnC: Ca2+ and 1:3 TnC: Ca2+. It is concluded that TnC existed in two conformational isomers that had different binding affinities for Ca2+: the binding of 3 Ca2+ was characteristic of a folded conformation (TnC A ) and the binding of 1 Ca2+ was characteristic of a partially unfolded conformation (TnCB). Both of these conformations contributed to the 8+ (and other) charge states of TnC, and were distinguished on the basis of their different Ca2+-binding affinities and not on the basis of the charge state. In the presence of the peptide, a complex with 1:1: 1 TnC: peptide: Ca2+ stoichiometry was formed.

[29] Use of differently immobilized nucleotides for binding NAD+-dependent dehydrogenases

Publisher Summary This chapter discusses the use of differently immobilized nucleotides for binding NAD + -dependent dehydrogenases. Structural analysis of several dehydrogenase enzymes has revealed the occurrence of a common, topologically similar, nucleotide binding domain. However, despite this structural homology, the nuances of enzyme specificity have necessitated the synthesis of an array of immobilized nucleotides for the purification of dehydrogenases by affinity chromatography. This is partly because the precise mode of binding of the nucleotide to its complementary site on the enzyme is often unknown and governs whether to design a nucleotide adsorbent, with an unsubstituted base, sugar, or phosphate grouping, and partly because the chemistry of immobilization is determined to a large extent by the convenience and available chemical protocols. This chapter discusses various positions on adenine nucleotides that can be readily derivatized to provide a spacer molecule suitable for the attachment to an insoluble support matrix. It reviews the preparation of several immobilized nucleotide derivatives and suggests the various nucleotide affinity matrices that might be preferable for a particular separation or purification. The chapter also recommends conditions for the adsorption and subsequent selective desorption of dehydrogenases from immobilized nucleotides to achieve optimal purifications or separations.