Biomedical subjects
Publications and source records attributed to R S Goody.
Present evidence on crossbridge behaviour is reviewed from the viewpoint of the Eisenberg-Hill concepts. We conclude that a cartoon illustrating the operation of these ideas in practice should be drawn so as to include morphological changes in the body of the myosin molecule as part of the power-stroke. The bulk of structural evidence on the operation of crossbridges in intact muscle supports such a description.
p21 isolated under nondenaturing conditions is obtained as a complex with guanosine nucleotides and magnesium ions. We have developed a high performance liquid chromatography method which removes greater than 95% of bound nucleotide and the metal ion very rapidly under mild conditions. At the same time, p21 is purified from minor protein impurities. The protein thus prepared is thermally much less stable than the complexed p21, but can be used for studying its interaction with nucleotides and metal ions at low temperatures. The association rate constant for p21 and GDP is 1.47 X 10(6) M-1 s-1 and for GTP is 2.9 X 10(6) M-1 s-1 at 0 degree C. By using appropriately determined dissociation rate constants we have determined the binding constant for p21.GDP and p21.GTP in the presence of excess Mg2+ to be 5.7 X 10(10) M-1 and 6.0 X 10(10) M-1, respectively, at 0 degree C.
Kinetic studies on the interaction of three Ha-ras-encoded p21 proteins with GDP and MgGDP have yielded values for the association (10(6)-10(7) M-1 s-1) and dissociation (10(-3)-10(-5) s-1) rate constants at 0 degrees C. Dramatic differences in the rate constants were not observed for the three proteins. Under non-physiological conditions (absence of Mg2+), the rate constant for GDP release was an order of magnitude faster for the viral protein p21v than for the cellular form p21c or the T24 mutant p21t, but this was reduced to a factor of about 3 in the presence of Mg2+. In all cases, there was an increase of about one order of magnitude in the rate of GDP release on removing magnesium. The binding affinities ranged from 5.7 X 10(10) M-1 for p21c to 1.3 X 10(11) M-1 for p21v. Electron paramagnetic resonance (EPR) measurements on Mn2+ bound together with stereospecifically 17O-labelled GDP showed direct coordination of a beta-phosphate oxygen to the metal ion with a superhyperfine coupling constant of 0.16-0.22 mT, but no interaction with the alpha-phosphate oxygens at the active site of all three proteins. The association constant of Mn(II) to p21 proteins in the absence of nucleotides was estimated to be greater than 10(5) M-1. In agreement with the EPR results, experiments on the metal ion dependence of the binding of thiophosphate analogs of GDP provided further evidence for the absence of direct coordination of the metal ion to the alpha-phosphate group. These results have been used to construct a model for the interactions of Mg X GDP with the active site of p21 proteins.
Glycerinated insect (Lethocerus) flight muscle in the presence of the non-hydrolysable ATP-analogue AMPPNP (1 mM at 4 degrees C) has been prepared for electron microscopy using X-ray diffraction monitoring during fixation and embedding. Superior preservation of the original structure has been achieved through use of a fixative which included tannic acid and excess Mg2+. New features have been recognized in single filament layers (myac and actin) and 15 nm cross sections. As previously shown, some aspects of relaxed structure (14.5 nm shelves along thick filaments) and of rigor (38.7 nm angled bridges along thin filaments) are retained in a modified form. New observations include: (1) In 15 nm cross sections that show single 14.5 nm levels: (a) The flared X structure characteristic of rigor is replaced by a straight-X figure in which the crossbridge density is aligned along the myosin-actin plane, rather than skewed across it as in rigor. (b) In AMPPNP, each crossbridge appears to have a separate origin from the thick filament, rather than bifurcating two from one stem as in the flared X of rigor. The separation of crossbridge origins is also evidenced by the loss of 'ladder rungs' in actin layers. (2) A halving (19.3 nm) of the 38.7 nm axial repeat along actin, rather than a thirding (12.9 nm) as in rigor, indicates redistribution of bridge attachments in cold AMPPNP. (3) In AMPPNP, the 14.5 nm shelves of density around the thick filament shaft are thicker but extend to smaller radius than similar shelves in ATP-relaxed muscle. This is shown by a lack of 14.5 nm periodicity and diffraction in actin layers of AMPPNP, in contrast to ATP-relaxed actin layers, in which the 14.5 nm period is present. Our results suggest that attached crossbridges are modified by AMPPNP and that ordered features of the analogue state are not accounted for solely by detached myosin heads or by a mixture of relaxed and rigor crossbridges. A two-domain model for the crossbridge is proposed. Domain 1 binds to the thin filament, and while bound, maintains a constant stereospecificity to actin at low resolution, independent of the type or presence of nucleotide at the myosin active site. Domain 2 is proximal to the thick filament and can exist in two characteristic states. In the absence of nucleotide (rigor), Domain 2 adopts a variable relationship to the thick filament, to accommodate the actin-bound end of the bridge.(ABSTRACT TRUNCATED AT 400 WORDS)
The mechanical events following release of ATP from P3-1-(2-nitro)phenylethyladenosine-5'-triphosphate (caged-ATP) in skinned guinea pig taenia coli smooth muscle in rigor were investigated. A rigor force of about 25-35% of the maximal active force was obtained by removing ATP at the plateau of a maximal active contraction. In the rigor solution free-Mg2+ was 2 mM, ionic strength 90 mM and pH 7.0. When caged-ATP (12.5 mM) was diffused into the preparation there was no change in the rigor force. Photolytic production of about 2 mM ATP was achieved with a xenon flash lamp. Following illumination, force decreased with an approximate initial rate constant of 0.7 s-1. The rate of relaxation was increased in the presence of inorganic phosphate (at 3 mM: 1.3 s-1; 10 mM: 2.2 s-1). At higher Mg2+ concentrations the rate of relaxation was slower (5 mM: 0.2 s-1) and at lower concentrations the rate was faster (0.5 mM: 1.2 s-1). An increased rate of relaxation was observed when ionic strength was increased to 150 mM (2.2 s-1). Phosphate increased the rate of relaxation at the different levels of Mg2+ (0.5-10 mM) and ionic strength (90, 150 mM). In preparations shortened (by 1-3%) to give reduced rigor force, a small transient increase in tension was recorded after ATP release. In comparison to the rates of ATP-induced dissociation of actomyosin in solution, reported in the literature, the rate of relaxation from rigor is slower. This may reflect a slow rigor cross-bridge dissociation or mechanical interactions not associated with cross-bridges in the muscle fibre.(ABSTRACT TRUNCATED AT 250 WORDS)
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v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.
A new procedure for determining DNA nucleotide sequences is reported. In the first step of the method, four DNAs, each separately substituted with a different deoxynucleoside phosphorothioate in place of the corresponding monophosphate, are prepared by template-directed polymerization catalyzed by DNA polymerase. In the second step, these DNAs are subjected to stringent exonuclease III treatment, which produces only fragments terminating with a phosphorothioate internucleotide linkage. These can then be separated by standard gel electrophoresis techniques and the sequence can be read directly as in presently used sequencing methods.
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The interactions of nucleotides with phosphoenolpyruvate carboxykinase were studied by using the stereospecific thiophosphate analogues of GDP and GTP. The metal ion dependent stereoselectivity of these analogues was determined by using steady-state kinetics. The RP and SP isomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S) were substrates with low turnover, and a small preference for the RP isomer was observed. Neither the enzyme-metal nor the nucleotide-metal complex elicited any substantial change in the selectivity. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) exhibited no substrate activity for the enzyme, regardless of the cations. This nucleotide was a competitive inhibitor against GDP, however. Both RP and SP diastereomers of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) were good substrates for phosphoenolpyruvate carboxykinase; in several cases, depending upon the cation, kcat and/or Vm/Km for the RP isomer is greater than for the substrate GTP. The enzyme-metal complex but not the nucleotide-metal complex affects the relative Km and the Vmax values. In contrast, guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) (SP) is a much better substrate (greater than 50 times) than is GTP beta S (RP). The metal ions have little effect on the selectivity. These results suggest a specific interaction of the beta-phosphate of the nucleotide with the protein. The analogue guanosine 5'-O-(3-thiotriphosphate) (GPT gamma S) serves as a substrate to yield GDP and thiophosphoenolpyruvate. The latter was detected by 31P NMR and was shown to slowly hydrolyze to form phosphoenolpyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)
31P-NMR spectra were obtained from adenosine-5'-tetraphosphate under a variety of conditions and the four discernible resonances assigned. The pK-value of the metal-free compound was determined to be 6.4, the pK-value of the Mg2+ complex to be 5.3. The dissociation constant for the AP4 X Mg2+ complex was estimated to be 10(-4) M from the downfield shift of the resonances assigned to the gamma- and delta-phosphorus nuclei. The binding of a second metal ion can also be followed by NMR; the dissociation constant for this ternary complex is several orders of magnitude larger than that for the binary complex.
The binding of rabbit muscle myosin subfragment 1 (S1) to glycerinated insect flight muscle fibers has been studied by low-angle x-ray diffraction, quantitative sodium dodecyl sulfate gel electrophoresis, quantitative interference microscopy, and electron microscopy. Changes induced in the rigor x-ray diffraction pattern are consistent with the idea that vacant myosin-binding sites on thin filaments are filled by exogenous S1. Electron microscopy indicates that S1 permeates and labels fibers and fibrils completely. Electron micrographs also show that cross-bridges are not displaced by exogenous S1 under the conditions used, and this is supported by the unchanged mechanical stiffness of the S1-labeled fibers. The amount of bound S1, as measured by gel electrophoresis and interference microscopy, together with the magnitude of the intensity changes in the x-ray diffraction pattern, is consistent with a thick filament structure that contains four molecules of endogenous myosin per 14.5 nm of its length, but does not agree well with earlier estimates of six myosins per crown. Lack of information on possible inhibition of S1-binding by factors other than the presence of cross-bridges, e.g., troponin, render uncertain calculations of the number of attached cross-bridges in the rigor state. However, it appears that at least 75% of the endogenous myosin heads are attached. Occupancy of binding sites on thin filaments after incubation with S1 is high, probably greater than 85%, so that x-ray scattering from those parts of the structure that adhere to the symmetry of the thin filaments can be treated as diffraction from S1-decorated thin filaments. In addition, we show in thin flared X cross sections that exo-S1 heads bind to actin with the geometry described in decorated actin by Taylor, K.A., and L.A. Amos.
The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.
Changes in the coordination of Mn2+ to nucleotide, water and protein at the active site of elongation factor Tu (EF-Tu) have been studied by electron paramagnetic resonance (EPR) spectroscopy. From the time dependence of the Mn2+ spectrum after addition of GTP to EF-Tu X Mn, it was apparent that three complexes with different EPR linewidths could be detected. Using additional information from the kinetics of 32Pi production and release from EF-Tu X Mn X [gamma-32P]GTP these were identified as EF-Tu X Mn X GTP (linewidth 4.2 mT), EF-Tu X Mn X GDP X Pi (1.20 mT) and EF-Tu X Mn X GDP (1.29 mT). The linewidth for EF-Tu X Mn was 1.51 mT. The rate constant for GTP cleavage on EF-Tu was 0.01 min-1 at 24 C, for Pi release from the EF-Tu X GDP X Pi complex 0.0033 min-1. The corresponding rate constants in the presence of Mg2+ were 0.003 min-1 and 0.0065 min-1. The rate constant for reversal of the cleavage step was found to be much smaller than that for the rate of Pi release (and consequently much smaller than in the forward direction), as shown by 31P-NMR experiments on the incorporation of 18O into Pi from GTP hydrolyzed in the presence of H2 18O. EPR experiments using specifically 17O-labelled GTPs demonstrated an interaction of Mn2+ with the beta-phosphate in both the EF-Tu X GDP X Pi and EF-Tu X GDP complexes. Inorganic phosphate in the EF-Tu X GDP X Pi complex was found not to interact with the metal ion. From EPR experiments in H2 17O, it was concluded that the most probable number of water molecules in the different complexes was 4 (EF-Tu X Mn), 5 (EF-Tu X Mn X GDP X Pi) and 3 (EF-Tu X Mn X GDP), with 2, 0 and 2 metal-protein interactions respectively.
Evidence from sequence studies and from proteolysis suggests that S1 consists of three domains. Cross-linking studies show that one S1 can bind to two actin monomers which may lie in different strands of the actin long helix. The S1-actin interaction comprises two states "weak" and "strong". We suggest there are distinct hinged binding sites, "weak" and "rigor", of which only the rigor site is sensitive to tropomyosin control. If one takes the weak binding domain to be a "nose-cone" which is attached to the rest of the S1 by a flexible covalent hinge allowing the rigor link to be formed independently a number of structural phenomena observed in fibres may be explained.
Recent experiments on the kinetics of the interaction between myosin subfragment 1 (S1) and F-actin in solution are summarized. It is concluded that, at every step of the ATPase cycle, the association between the two proteins takes place in two stages. The equilibrium constant of the second step and thus the affinity of S1 for actin changes from step to step during the enzymatic reaction. It is proposed that the transient kinetic evidence can be interpreted in terms of two different classes of contraction models. The first one, which is widely used at present, identifies particular steps in the enzymatic reaction as directly responsible for the conformational change which represents the power stroke of muscle contraction (direct coupling model). In the second class of model, to which we wish to draw attention, changes in affinity modulated by the enzymatic reaction result in changes in the relative amounts of time spent by parts of the myosin molecule in two different environments. These environments determine whether the molecule exists in the 'long' or 'short' state, and it is the transition between these two which constitutes the power stroke (indirect coupling model).
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The geometry of the Me2+. GTP complex at the active site of EF-Tu from Bacillus stearothermophilus has been investigated using thiophosphate analogs of GTP to inhibit the kirromycin-induced GTPase reaction at 60 mM NH4Cl. There is no reversed selectivity for the diastereomers (Rp and Sp) of guanosine 5'-O-(1-thiotriphosphate) (GTP[alpha S]) on replacing Mg2+ by Cd2+, so that the observed specifity for the Sp isomer must be due to an interaction of the pro-R oxygen of the alpha-phosphate group with the protein. With the diastereomers of GTP[beta S] low specifity for the Rp isomers is seen in the presence of Mg2+. Moreover, both isomers are very weakly bound. In contrast, substitution of Mg2+ by Cd2+ results in a high specifity for the Sp isomer, and this is then recognized as well as Cd X GTP. These results indicate that in the EF-Tu X Me2+ X GTP complex, the pro-S oxygen of the beta-phosphate group is bound to the metal ion and the pro-R oxygen to the protein. GTP[gamma S] is a good analog of GTP regardless of the nature of the metal ion, suggesting that not all of the oxygens of the gamma-phosphate are involved in interactions to metal ion and protein. The thiophosphate analogs of GTP were also tested for their efficiency in ternary complex formation with EF-Tu and aminoacyl-tRNA and in the physiological GTPase of EF-Tu. The stereochemistry of the GTP binding site on EF-Tu in all three systems is found to be very similar.