The Amino Acid Sequence of a Hexapeptide Containing an Essential Sulfhydryl Group of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase<sup>*</sup>

Gold, Alvin H.; Segal, Harold L.

doi:10.1021/bi00894a008

Cited by 36 publications

(7 citation statements)

References 23 publications

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“…The shape of the spectrum for SF,-DDPM is essentially identical with that of DDPM reacted with /3-mercaptoethanol in Tris-KCl buffer, pH 8.0, indicating the protein does not affect the DDPM chromophore significantly. A molar absorptivity of 2930 M'1 cm'1 at 442 nm was determined for the DDPM-jS-mercaptoethanol adduct, in good agreement with the value obtained previously by Gold & Segal (1964). When the DDPM-f)-mercaptoethanol spectrum and the corrected emission spectrum of AEDANS-SF | were used, a spectral overlap integral (/) of 6.1 x 10'3 M"1 cm'1 nm4 was determined.2 2 During the course of this work, it was observed that the spectrum of SF,-DDPM changed as a function of time due to base-promoted hydrolysis of the maieimide ring (Yamamoto et al, 1977).…”

Section: Resultssupporting

confidence: 89%

“…The 280-nm absorption of AEDANS labeled stoichiometrically to SF, was less than 1.5% of that for the protein. The amount of DDPM bound to SF, was determined by the absorption at 442 nm by using <z = 2930 M'1 cm'1 (Gold & Segal, 1964). Protein concentration of DDPM-modified SF, was determined by a dye binding method (see below) since DDPM has significant absorption at 280 nm.…”

Section: Methodsmentioning

confidence: 99%

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Foerster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1

Dalbey¹,

Weiel²,

Yount³

1983

Biochemistry

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Förster energy transfer was used to measure the distance between reporter groups on the two reactive thiols of myosin, SH1 and SH2, and to detect changes in this distance upon binding of nucleotide. SH1 was labeled with the fluorophore 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (1,5-IAEDANS) and SH2 with the chromophoric acceptor N-[4-(dimethylamino)-3,5-dinitrophenyl]-maleimide (DDPM). Peptide studies verified that [3H]-1,5-IAEDANS reacted specifically with SH1, while [14C]DDPM labeled both SH2 and the alkali light chains. The [14C]-DDPM-modified alkali light chains were replaced with unmodified light chains by the exchange procedure of Wagner and Weeds [Wagner, P.D., & Weeds, A. G. (1977) J. Mol. Biol. 109, 455-473]. Subfragment 1 labeled with 1,5-IAEDANS and then with DDPM exhibited two fluorescence lifetimes, 20.6 (AEDANS-SF1, unquenched) and 9.3 ns (AEDANS-SF1, quenched by DDPM). The latter lifetime decreased to an average of 2.85 ns after the addition of MgAMP-PNP, MgADP, or MgPPi (no change with MgAMP), indicating that the distance between the donor and acceptor decreased. An R0 of 29 A was calculated for the AEDANS/DDPM system assuming random orientation of the donor/acceptor pair. The decrease in the observed lifetimes upon the addition of Mg nucleotide corresponds to a change in the donor-acceptor distance from 28 to 21-22 A. This observation is consistent with the proposal that nucleotide binding juxtaposes SH1 and SH2 to enhance their cross-linking with various bifunctional reagents [Burke, M., & Reisler, E. (1977) Biochemistry 16, 5559-5563; Wells, J. A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970].

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Section: Resultssupporting

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Foerster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1

Dalbey¹,

Weiel²,

Yount³

1983

Biochemistry

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“…1,5-IAEDANS does not absorb at 442 nm and therefore does not interfere with optical density measurements at that wavelength. The extinction coefficient determined for DDP was 2930 M Ϫ1 cm Ϫ1 at 442 nm, in good agreement with the value reported by Gold and Segal (1964), and 3100 M Ϫ1 cm Ϫ1 at 337 nm. Using these extinction coefficients and the Bradford protein determination assay, the stoichiometry of labeling was determined for each of the singly and doubly labeled RLC mutants.…”

supporting

confidence: 90%

Proximity Relationships between Engineered Cysteine Residues in Chicken Skeletal Myosin Regulatory Light Chain:

Wolff-Long

Tao

Lowey

1995

Journal of Biological Chemistry

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Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys 2 and Cys 155and Cys 73 and Cys 155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. The fluorescent and nonfluorescent probes N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used as the donor and the acceptor, respectively. The distance between Cys 2 and Cys 155 was measured to be 35 and 30 Å in the absence and presence of myosin heavy chain, respectively, suggesting a slightly more compact structure for the light chain in the bound state. The distance between Cys 73 and Cys 155 measured in a similar manner was 31 and 30 Å in the free and bound states, respectively; this latter value is in good agreement with that derived from crystallographic structures. For heavy chain-bound light chains, no measurable distance changes were detected with the binding of ATP or actin. These results show that no gross structural changes occur within the regulatory light chain during the contraction cycle, but that resonance energy transfer between other sites could be used to monitor potential changes in the myosin head upon the binding of nucleotides and actin.Force generation in muscle results from a cyclic interaction of the myosin head (S1) 1 with actin. It is currently believed that S1 may change its orientation relative to the actin filament during this process and thereby produce a displacement of ϳ50 -100 Å, the so-called "working stroke" (Huxley, 1969;Huxley and Simmons, 1971). A persistent problem with the "rotating cross-bridge model" has been the failure to detect any large conformational change in the head by most biophysical methods. It has been suggested that the reactive Cys 707 on the heavy chain, the site of labeling for most spectroscopic probes, may not be particularly sensitive to angular changes, and the portion of S1 adjacent to the rod might undergo more pronounced structural changes during hydrolysis (Huxley and Kress, 1985). We have therefore focused our efforts over the last few years on preparing Cys mutants of the regulatory light chain (RLC), a subunit which is readily exchanged into myosin, and can therefore provide a means to introduce probes into the COOH-terminal region of the myosin head (Saraswat and Lowey, 1991;Saraswat et al., 1992). Moreover, this approach avoids modification of the reactive heavy chain Cys residues, SH1 and SH2, which can have adverse effects on the enzymatic activity and motor properties of myosin (Root and Reisler, 1992).Several earlier studies have used labeled light chains to detect structural changes in the myosin head. By incorporating a 1,5-IAEDANS-labeled RLC into smooth muscle myosin, Morita et al. (1991) were able to show by fluorescence anisotropy that a change in conformation at the head/rod junction can be correlated with the transition from a folded to an extended myosin molecule. Hambly et al. (1991) exchanged a spin-labeled light chain into skinned skeletal muscle ...

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“…The modified actin was dialysed against G buffer, clarified by centrifugation at 100 000 g for 45 min and filtered through a Sephadex G-25 column. The labelling ratio was calculated using a molar absorption coefficient of 3.05i10$ M −" :cm −" at 440 nm [30]. The labelling ratio of five independent preparations was between 0.50 and 0.93.…”

Section: Labelling Of Actin With DC and Ddpmmentioning

confidence: 99%

Structural changes in subdomain 2 of G-actin observed by fluorescence spectroscopy

Moraczewska

Strzelecka‐Gołaszewska

Moens

et al. 1996

Biochemical Journal

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The influence of DNase I binding to Ca-ATP-G-actin and of Ca2+/Mg2+ and ATP/ADP exchange on the conformation of G-actin were investigated by measuring the fluorescence of dansyl cadaverine (DC) conjugated to Gln41 in subdomain 2 of the protein. Fluorescence resonance energy transfer (FRET) between this probe and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM) attached to Cys374 in subdomain 1 was also measured. Contrary to an earlier report [dos Remedios, Kiessling and Hambly (1994) in Synchrotron Radiation in the Biosciences (Chance, B., Deisenhofer, J., Ebashi, S., Goodhead, D. T., Helliwell, J. R., Huxley, H. E., Iizuka, T., Kirz, J., Mitsui, T., Rubenstein, E. et al., eds.), pp. 418-425, Oxford University Press, Oxford], the distance between these probes did not change significantly when DNase I was bound to actin. A small but reproducible increase in the quantum yield and a blue shift of the DC fluorescence maximum were observed when bound Ca2+ was replaced by Mg2+. A large increase (about 70%) in the quantum yield and an approx. 12 nm blue shift of the emission spectrum occurred when ATP in Mg-G-actin was replaced by ADP. These changes were not accompanied by any significant change in the FRET distance between the dansyl donor and DDPM acceptor probes. A substantial change in the fluorescence of DC-actin was observed after proteolytic removal of the last three residues of actin, in accordance with earlier evidence suggesting that there is a conformational coupling between subdomain 2 and the C-terminal segment in subdomain 1 of actin. The results are discussed in relation to recently published data obtained with another fluorescent probe and to earlier observations based on limited cleavage using proteolytic enzymes.

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The Amino Acid Sequence of a Hexapeptide Containing an Essential Sulfhydryl Group of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase^*

Cited by 36 publications

References 23 publications

Foerster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1

Foerster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1

Proximity Relationships between Engineered Cysteine Residues in Chicken Skeletal Myosin Regulatory Light Chain:

Structural changes in subdomain 2 of G-actin observed by fluorescence spectroscopy

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The Amino Acid Sequence of a Hexapeptide Containing an Essential Sulfhydryl Group of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase*

Cited by 36 publications

References 23 publications

Foerster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1

Foerster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1

Proximity Relationships between Engineered Cysteine Residues in Chicken Skeletal Myosin Regulatory Light Chain:

Structural changes in subdomain 2 of G-actin observed by fluorescence spectroscopy

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About

The Amino Acid Sequence of a Hexapeptide Containing an Essential Sulfhydryl Group of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase^*