On the tryptophan residue of smooth muscle myosin that responds to binding of nucleotide

Abstract

D iscoveries that on binding MgATP (or MgADP) the intrinsic UV absorbance (1) and fluorescence intensity (2) of myosin changes have enabled the quantitative-albeit empirical-study of myosin ATPase kinetics (3, 4). Earlier studies have attempted to distinguish between various classes of Trp and to determine the nature and identity of the Trp residues, whose fluorescence is most influenced on addition of nucleotide (5, 6). However, it has not been known which tryptophan residue [there are seven for skeletal muscle (7) or 10 for smooth muscle (8) in the unitary myosin fragment, subfragment 1] responds on binding. During the last decade there have been reports (9, 10) that the ATP-sensitive tryptophan residue in rabbit skeletal myosin is Trp-512**, but recently, Reshetnyak et al. † † claimed that it is Trp-441. By combining site-directed mutation of Dictyostelium with homology, Batra and Manstein (12) find that it is Trp-512. As our present paper was being readied for publication, Yengo et al. ‡ ‡ , using a ''one-Trp-only'' strategy in mutating smooth muscle myosin, more directly confirm Trp-512. In this paper, using single Phe substitution of either Trp-441 or Trp-512, we, too, document that Trp-512 is the responsive one and are aware that, in a novel spectroscopic approach, Park and Burghardt § § reached the same conclusion. Thus the controversy seems to us ended. Fortuitously, however, our ''one substitution at a time'' strategy has led us to find interesting additional findings. It turns out that the mutant W512F heavy meromyosin (HMM) (constructed originally to contrast with wild-type HMM) has ATPase properties strikingly like those of wild-type HMM that has been chemically reacted with a common thiol reagent. From this fact, we infer that changes at position 512 are transmitted to position 717, where they cause the same changes as are alternatively caused by reacting Cys-717 with thiol reagent. The changes at Cys-717, caused either way, are, in turn, transmitted to the active site. We say that there is a ''path of influence'' (15) extending from 512, to 717, to the active site. Some details about this path are given below. Materials and MethodsProtein Preparations. Rabbit skeletal muscle actin was purified by the method of Spudich and Watt (16). Chicken gizzard myosin light chain kinase and bovine testis calmodulin were prepared as described by Adelstein and Klee (17) and Yazawa et al. (18), respectively.Construction of Recombinant Baculoviruses. cDNA constructs for wild-type and two mutant (W441F and W512F) HMM heavy chains linked to a His tag (at the N terminus) and a myc tag (at the C terminus) were prepared as described in Kojima et al. (19). Briefly, GMH-6, a cDNA encoding the N-terminal half (Met-1 to Glu-729) of the chicken gizzard HMM heavy chain (8), was mutagenized according to the method of Kunkel et al. (20) with two oligonucleotides: 5Ј-TTTGAACGTCTCTTCCGTTTCAT-TCTAACTCGTGTAAAC-3Ј to replace Trp-441 with Phe and 5Ј-CAGCGTGAGGGCAT TGA ATT TA AT T TCAT TGA-CTTTGGCC-3Ј to replace Trp-512 with Phe. Th...

Dynamics of actomyosin interactions in relation to the cross-bridge cycle

“…There is now abundant evidence that this signal arises from a conserved tryptophan residue (Dd W501 Malnasi-Csizmadia et al 2000), skeletal W510 (Park & Burghardt 2000) and smooth myosin W512 (Onishi et al 2000;Yengo et al 2000)). By using a single tryptophan construct (i.e.…”

Early stages of energy transduction by myosin: Roles of Arg in Switch I, of Glu in Switch II, and of the salt-bridge between them

“…AcNPV͞ELC͞RLC viruses (for essential and regulatory light chains) were prepared (11). Expression and purification of recombinant HMMs were carried out (7,12), except that affinity-purified His-tagged HMMs were further purified by chromatography using a Pharmacia MonoQ column. To remove bound nucleotides from mutant HMMs, the preparation was dialyzed for 2 days against 500 ml of buffer, 0.45 M KCl, 1 mM EDTA, 20 mM Tris⅐HCl (pH 7.5), and 0.5 mM DTT, on ice with four-time buffer changes.…”

“…Several studies (12,(15)(16)(17) have suggested that Trp-512 (of smooth muscle myosin) is the responsible Trp residue. Crystallographic studies demonstrated that the ATP-induced rotation of the lower piece of 50 kDa, which bears Trp-512 at its tip, can perturb this fluorophore (5).…”

“…In this work, we take a rapid increase in fluorescence emission from HMM as evidence that, on binding at the active site, an influence quickly travels from the site to a particular residue, Trp-512 (12,(15)(16)(17). In the case in which a mant fluorophore has access to the active site, under illumination at 290 nm, we take a rapid increase in fluorescence at 455 nm as evidence that the fluorophore is more or less statically bound to the active site (assuming that such a fluorophore is tuned only to accept energy from Trp and then re-emit it at 455 nm), but we do not claim to know the identity of the originating Trp.…”

An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

“…W506F retains nucleotide sensitive fluorescence enhancement despite significant perturbation of ATPase functionality. W5061(Y499F) also shows a perturbed ATPase functionality common for side-chain modifications in the vicinity of W506 (46,47).…”