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M F Morales

Publications and source records attributed to M F Morales.

At least 19 recordsLinked to original sources
PubMed1992

The sequence location of the actin metal.

We have incorporated Fe2+ into the high-affinity metal-ion-binding site of actin. By supplying the system with oxygen from air and a reductant (dithiothreitol or ascorbate), we have induced free-radical generation, with the intent of causing peptide cleavage at the metal-ion-binding site. By analysis of the resulting fragments from actin in the F-form, we have deduced that cuts occurred at positions 159-160 and 301-302 (at the latter location we could not be sure if more than one cut occurred). We considered that these two cuts occurred in the chain strand coursing from the outer to the inner domain and vice-versa. Our results harmonize very well with the recently reported atomic structure of actin [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F. & Holmes, K.C. (1990) Nature 347, 37-44] and remove ambiguities that had remained in the structure. The results partly bear out the homology-based prediction of Strzelecka-Golaszewska et al. [Strzelecka-Golaszewska, H., Boguta, G., Zmorzynshi, S. & Moraczcwska, J. (1989) Eur. J. Biochem. 182, 299-305].

Actins↗
PubMed1991

A search for protein structural changes accompanying the contractile interaction.

It appears that small movements (detected hitherto only by fluorescence resonance energy transfer measurements and crosslinking studies) in a region of the myosin S-1 particle may mediate chemomechanical energy transduction in the contractile system. Here we find under conditions of high precision at 10 degrees C and 20 degrees C that ATP binding to S-1 causes small (0.4%) changes in CD signal, delta epsilon 222, as do temperature changes in the regime below 16 degrees C. ATP binding perturbs tryptophan residues that we now think are in the mobile region, and we find here that temperature affects tryptophan fluorescence in much the same way that it affects the CD signal, so we believe that the CD signal reports transduction-related movements in S-1. If S-1 is exposed to the range 16-30 degrees C, CD signal falls with temperature; ATP counteracts this fall. Analysis of vacuum-UV CD spectra yields 42% alpha-helix, 9% antiparallel beta-sheet, 7% parallel beta-sheet, 14% beta-turns, and 29% other structures.

Adenosine Triphosphate↗
PubMed1990

Nucleotide regulation of vasoactive intestinal peptide binding to bovine thyroid plasma membranes.

The specific binding of vasoactive intestinal peptide (VIP) to bovine thyroid plasma membranes is inhibited by guanine nucleotides. Guanosine 5'-triphosphate (GTP) and the non-hydrolyzable GTP analogs guanosine 5'-beta,gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) inhibited markedly the binding of VIP to its receptors. This inhibition was higher with GTP than with Gpp(NH)p and GTP-gamma-S and was due to an increase of the rate of dissociation of peptide bound to membranes. Other nucleotides did not show any effect.

Animals↗
PubMed1989

Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex.

Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near the actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1), to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido)fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5'-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide or N-[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the fluorescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 A; Cys-697 (S1), 49.4 A; active site of S1, greater than or equal to 44 A; nucleotide binding site (actin), 41.1 A; and Cys-374 (actin), approximately 53 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1989

Effect of chronic intake of ethanol on the binding of vasoactive intestinal peptide to rat spleen lymphoid cells.

1. The effect of chronic intake of ethanol on the binding of vasoactive intestinal peptide (VIP) to rat spleen lymphoid cells was investigated. 2. The intake chronic of ethanol elicited an increase in specific VIP binding. 3. This increase was due to an increase in binding capacity of both the high and the low affinity binding sites. 4. There was a decrease in the affinities of both classes of VIP binding sites.

Alcoholism↗
PubMed1989

On the origin and transmission of force in actomyosin subfragment 1.

A proximity map showing the three-dimensional arrangement of 12 chemically defined points in actomyosin subfragment 1 is developed and roughly correlated with published electron microscope reconstruction of others. Several additional points and topological relationships in the primary polypeptide chain folding are assimilated into this model. Certain crosslinkings and distance change observations are interpreted as indicators of transmission of force/displacement between the nucleotide-binding and an actin-binding site--i.e., as indications of how energy is transduced in this system.

Microscopy, Electron↗
PubMed1988

Orientation of actin monomer in the F-actin filament: radial coordinate of glutamine-41 and effect of myosin subfragment 1 binding on the monomer orientation.

We have employed the method of radial distance measurements in order to orient the actin monomer in the F-actin filament. This method utilizes fluorescence resonance energy transfer measurements of the distance between two equivalent chemical points located on two different monomers. The interprobe distance obtained this way is used to compute the radial coordinate of the labeled amino acid [Taylor, D. L., Reidler, J., Spudich, J. A., & Stryer, L. (1981) J. Cell Biol. 89, 362-367]. Theoretical analysis has indicated that if radial coordinates of four points are determined and six intramolecular distances are known, one can, within symmetry limits, position the monomer about the filament axis. The radial distance of Gln-41 that had been enzymatically modified with dansyl, rhodamine, and fluorescein derivatives of cadaverine was found to be approximately 40-42 A. The determination of the radial distance of Cys-374 was accomplished by using monobromobimane and N-[[(iodoacetyl)amino]ethyl]-5- naphthylamine-1-sulfonate as donors and N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide as acceptor; the results were consistent with a radial coordinate for this residue of 20-25 A. The effect of myosin subfragment 1 (S1) binding on the radial coordinates of (1) Gln-41, (2) Cys-374, and (3) the nucleotide binding site was also examined. S1 had a small effect on the radial coordinate of Gln-41, increasing it to 44-47 A. In the two remaining lases the change in the radial coordinate due to the S1 binding was negligible. This finding excludes certain models of the interaction between actin and S1 in which actin monomer rotates by a large angle when subfragment 1 binds to it.

Actins↗
PubMed1988

The myosin SH2-50-kilodalton fragment cross-link: location and consequences.

Some of us recently described a new interthiol cross-link which occurs in the skeletal myosin subfragment 1-MgADP complex between the reactive sulfhydryl group "SH2" (Cys-697) and a thiol (named SH chi) of the 50-kilodalton (kDa) central domain of the heavy chain; this link leads to the entrapment of the nucleotide at the active site [Chaussepied, P., Mornet, D., & Kassab, R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2037-2041]. In the present study, we identify SH chi as Cys-540 of the 50-kDa fragment. The portion of the heavy chain including this residue and also extending to Cys-522 that is cross-linkable to the "SH1" thiol [Ue, K. (1987) Biochemistry 26, 1889-1894] is near the SH2-SH1 region. Furthermore, various spectral and enzymatic properties of the (Cys697-Cys540)-N,N'-p-phenylenedimaleimide (pPDM)-cross-linked myosin chymotryptic subfragment 1 (S-1) were established and compared to those for the well-known (SH1-SH2)-pPDM-cross-linked S-1. The circular dichroism spectra of the new derivative were similar to those of native S-1 complexed to MgADP. At 15 mM ionic strength, (Cys697-Cys540)-S-1 binds very strongly to unregulated actin (Ka = 7 X 10(6) M-1), and the actin binding is very weakly affected by ionic strength. Joining actin with the (Cys697-Cys540)-S-1 heavy chain, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, produces different species than does joining unmodified S-1 with actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1988

Modifying preselected sites on proteins: the stretch of residues 633-642 of the myosin heavy chain is part of the actin-binding site.

We have designed an "antipeptide" capable of firmly and specifically interacting with a preselected stretch of myosin S-1 heavy chain. Covalent attachment of this antipeptide to its target stretch, residues 633-642, does not affect the intrinsic ATPase activities of the protein but significantly reduces the actin-binding capabilities of the myosin head.

Actins↗
PubMed1988

The micromechanics of contraction.

This paper suggests the molecular mechanism in muscle whereby some of the delta G of ATP hydrolysis is converted to mechanical work. There is good evidence for thinking that all mechanical, thermal, and chemical observations of muscle result from the summated, repetitive, operation of identical unitary "engines". Such an engine is the S-1 moiety of a myosin crossbridge and the adjacent actin. The operational principle is evident on realizing that S-1 is a particle with two interactive ligand (ATP and actin) binding sites. The change resulting from binding nucleotide is propagated to the actin-binding site, there specifying affinity and angles of attachment. Repetition follows because stepwise enzymatic degradation through intermediates is equivalent to cyclical changing of the site occupant. Trans- (S-1) propagation ensures a work cycle because actin held at a succession of relative positions can generate external work. Proximity mapping and protein chemical studies of S-1 suggest that variation in the position at which actin is held results from the simultaneous operation of a continuing (S-1)-actin contact and a polyphosphate charge-modulated coulombic contact.

Actins↗
PubMed1987

Two kinds of transducer.

After recognizing that there are two kinds of biotransducers, attention is focused on the kind that transduces energy, as in muscle or in ion pumps. Using muscle as the example, it is suggested that the essential elements in the transducer (S-1 segment of myosin) are (1) An ATPase site that, in time, is occupied by a succesion of ligands ("intermediates"); (2) A spatially separate site for binding a second ligand (actin, in the case of muscle) that can be held in a variety of spatial relations; and (3) A transmitting mechanism (electric field, propagated structural distortion) that allows the conformation impressed by the ligand of (1) to be conducted over space and impose a specific relation to the ligand of (2).

Biophysical Phenomena↗
PubMed1986

Modification of the actin interface of skeletal myosin subfragment-1 by treatment with dibromobimane.

Recently, by treating the head portion of skeletal myosin subfragment-1 (S1) with the bifunctional agent dibromobimane, we introduced an intramolecular covalent cross-link which resulted in the stabilisation of an internal loop in the heavy chain structure of the head [Mornet et al. (1984) Proc. Natl Acad. Sci. USA 82, 1658-1662]. In order to define the functional properties of this new S1 conformational state, we have first determined the experimental conditions for the optimum modification of S1 by dibromobimane. We finally settled on a 60% yield of cross-linked S1. Because the modification occurs between the 50-kDa and the 20-kDa tryptic heavy chain fragments which have been postulated to be involved in the interaction of native S1 with actin, we have investigated the association of dibromobimane-treated S1 with actin, using chemical cross-linking of their rigor complex with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linked species obtained were analyzed by polyacrylamide gel electrophoresis and compared with those known for unmodified S1. The carbodiimide-catalyzed linkage between actin and dibromobimane-modified S1 led to a singlet protein band migrating with an apparent molecular mass of 155 kDa, in contrast to the usual doublet bands of 175 kDa and 185 kDa produced with native S1. This result suggests that a change has occurred at the actin interface on the dibromobimane-treated S1 heavy chain. The covalent complex generated by carbodiimide cross-linking between actin and dibromobimane-modified S1 (27-kDa + 50-kDa + 20-kDa fragments) was submitted to chemical hydrolysis with hydroxylamine. The nature of the products identified is consistent with the conclusion that the internal freezing of the heavy chain structure by dibromobimane induces the loss of the ability to cross-linkage of the actin site on the 20-kDa domain but does not affect the conformation of the second site on the 50-kDa segment, which becomes the unique actin region cross-linkable by actin.

Actins↗
PubMed1985

Exposure of actin thiols by the removal of tightly held calcium ions.

The removal of bound metal ions from G-actin uncovered two thiols, Cys-10 and Cys-257. The uncovering of these thiols requires a free calcium concentration lower than 10 nM. Therefore, participation of one or both thiols in Ca2+ binding is suggested. Actin labeled with N-(5-fluoresceinyl)maleimide in the absence of calcium moves as a doublet in NaDodSO4/PAGE. It is suggested that two conformers are induced by metal removal and labeling.

Actins↗
PubMed1985

Stabilization of a primary loop in myosin subfragment 1 with a fluorescent crosslinker.

A bifunctional fluorescent alkylating agent, dibromobimane, has been used to stabilize a preexisting primary loop in myosin subfragment 1 (S-1). The crosslink achieved joins Cys-707 (called sulfhydryl group "SH1") of the 20-kDa domain (formerly called "20K" domain) with a thiol of the 50-kDa domain and seems to place the dibromobimane near the ATP-perturbable tryptophan.

Animals↗
PubMed1984

Application of the Dale-Eisinger analysis to proximity mapping in the contractile system.

Fluorescence anisotropy decays were used to quantify the degree of rapid librational motion associated with several fluorescent probes attached to contractile proteins. This information allows an analysis of how far kappa 2, the resonance energy transfer orientation factor, may deviate from the dynamically averaged value of 2/3. Extrema can then be set on R0, the critical transfer distance, and hence on the interprobe distance. These results set maximum ranges on possible distances between several probe pairs used in the mapping of contractile protein structure by resonance energy transfer.

Actins↗
PubMed1984

Proteolysis and the domain organization of myosin subfragment 1.

Because the proteolytic cleavage of a folded polypeptide depends not only on the specificity of the protease but on the nature of the folding, we investigated the cleavage of (chymotryptically produced) subfragment 1 (designated "S-1") or "head" segment of myosin by seven proteases with different specificities. All seven produced approximately the same three fragments of S-1--namely, fragments (from the NH2 terminus) of 27, 50, and 20 kilodaltons, suggesting that in intact S-1 these fragments are distinct domains. The same proteases were used to hydrolyze the MgADP complex of S-1. All failed to do so except trypsin, which, as found earlier [Hozumi, T. (1983) Biochemistry 22, 799-804], makes two additional cleavages. This result suggests that the conformational change induced by MgADP opens up only a small stretch of polypeptide chain, which stretch happens to be vulnerable to trypsin.

Animals↗
PubMed1984

Isolation and partial renaturation of proteolytic fragments of the myosin head.

Methods have been devised for isolating two of the tryptic fragments (those termed "20K" and "50K") of myosin "subfragment 1" in pure form. Fragment 20K was examined for renaturation after removal of denaturants used in its preparation. It generated a CD spectrum corresponding to ca. 64% formed structure (roughly what would be expected from its amino acid sequence) and a red-shifted UV spectrum such as arises when phenylalanine and tyrosine are perturbed by structural interactions. Actin affinity of fragment 20K was tested by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking, inhibition of the actin-activated ATPase of subfragment 1 containing light chain 3, cosedimentation with actin, and light scattering; the affinity exceeded 5 X 10(6) M-1. The foregoing suggests that moiety 20K has a sovereign existence in (i.e., is a domain of) myosin subfragment 1. Preliminary work indicates that fragment 50K also binds actin, but with a lesser affinity.

Actins↗