Effect of Metal Cations on the Conformation of Myosin Subfragment-1-ADP-Phosphate Analog Complexes:  A Near-UV Circular Dichroism Study

Ym, Peyser; Ajtai, Katalin; Mm, Werber; Tp, Burghardt; Mühlrád, András

doi:10.1021/bi970255y

Cited by 21 publications

(35 citation statements)

References 27 publications

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“…Because of these somewhat conflicting results it is important to study how the various phosphate analogue (PA)‐containing complexes resemble the specific intermediates of the ATPase cycle in solution. The results of solution studies, including near UV CD measurements [10], kinetics of Cys‐707 [11] and Lys‐83 [12] modifications, mostly supported the conclusions of the results obtained with the recombinant truncated Dictyostelium S1 [7,8], i.e. S1·ADP·BeF x resembles the M * ATP, whereas S1·ADP·AlF 4 − and S1·ADP·V i mimic the M ** ADP·P i intermediate state.…”

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confidence: 60%

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

Peyser

Mühlrád

1999

European Journal of Biochemistry

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Biochemistry 34, 9500±9507] reported that 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin-binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the 1-amino group of Lys553 reacts with FHS. Addition of 0.4 m KCl reduced the rate of reaction significantly, which indicates ionic strengthdependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP´BeF x , ADP´AlF 4 , ADP´V i and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide-induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553-bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide-induced conformational change. In the presence of ATP when the M ** ADP´P i state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1´ADP´AlF 4 2 and S1´ADP´V i complexes, which are believed to mimic the M ** ADP.P i state. This indicates that the conformation of the S1±ADP´AlF 4 2 and S1´ADP´V i complexes in the vicinity of Lys553 does not resemble the structure of the M ** ADP´P i state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553-bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.

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confidence: 60%

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

Peyser

Mühlrád

1999

European Journal of Biochemistry

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“…CD Assay of Charge-Transfer Complexes-CD spectra were recorded in a 10-mm path length cuvette at 4°C on a Jasco J-720 (Easton, MD) (36,37). Enzyme was diluted in a quartz cuvette to a concentration of 5-15 M in 10 mM potassium phosphate buffer, pH 8.0, 10% glycerol; molecular oxygen was removed as above, and the CD spectrum was measured from 250 to 700 nm.…”

Section: Construction Of Sbcad Prokaryotic Expression Vectors-mentioning

confidence: 99%

“…4C). Such a difference between the absorbance and CD spectra suggests a difference in the conformational shift of the reduced form of enzyme when it complexed with different substrates (25,27,36,52). This could be due to alterations in the conformation of the flavin isoalloxazine ring itself or in its position relative to an amino acid residue that can alter the flavin signal.…”

Section: Kinetic Properties Of Purified Wild Type and Mutantmentioning

confidence: 99%

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Burghardt

Vockley

2003

Journal of Biological Chemistry

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Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to ␤-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.The acyl-CoA dehydrogenases (ACDs) 1 are a family of related enzymes that catalyze the ␣, ␤-dehydrogenation of acylCoA esters, transferring electrons to electron transferring flavoprotein (1-4). Deficiencies of these enzymes are important causes of human disease (3-7). Biochemical and immunological studies have identified at least 9 distinct members of this enzyme family, each having a characteristic substrate utilization pattern (8 -13). Very long, long, medium, and short chain acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively) catalyze the first step in the mitochondrial ␤-oxidation of fatty acids with substrate optima of 16, 16, 8, and 4 carbon chains, respectively (3, 14 -15). A new ACD has been identified recently (ACAD9) that is also active against long chain acyl-CoA substrates (16). Isovaleryl-CoA dehydrogenase (IVD), short/branched chain acyl-CoA dehydrogenase (SBCAD, also known as 2-methyl branched chain acyl-CoA dehydrogenase), and isobutyryl-CoA dehydrogenase (IBD) catalyze the third step in leucine, isoleucine, and valine metabolism, respectively (2-4, 11, 12, 17, 18), whereas glutaryl-CoA dehydrogenase is active in lysine metabolism (19).We have shown previously that SCAD, SBCAD, and IBD can all utilize butyryl-CoA as substrate (albeit with different efficiencies), whereas ...

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“…The MgATP hydrolysis is coupled to a series of conformational changes in myosin, resulting in a cycle of attachment to the actin filament, strain development, release, and re-attachment. Myosin structural intermediates from the cycle summarized in Scheme 1 were uniquely identified by near ultra-violet circular dichroism spectra and nucleotide sensitive tryptophan fluorescence intensity changes (3). …”

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Single Myosin Lever Arm Orientation in a Muscle Fiber Detected with Photoactivatable GFP

Burghardt

Ajtai

2009

Biochemistry

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Myosin 2 is the molecular motor in muscle. It binds actin and executes a power stroke by rotating its lever arm through an angle of ~70° to translate actin against resistive force. Myosin 2 has evolved to function optimally under crowded conditions where rates and equilibria of macromolecular reactions undergo major shifts relative to those measured in dilute solution. Hence an important research objective is to detect in situ the lever arm orientation. Single molecule measurements are preferred because they clarify ambiguities unavoidable with ensemble measurements, however, detecting single molecules in the condensed tissue medium where myosin concentration exceeds 100 µM is challenging. A photoactivatable green fluorescent protein (PAGFP) tagged myosin light chain (MLC) was constructed. The recombinant MLC physically and functionally replaced native MLC on the myosin lever arm in a permeabilized skeletal muscle fiber. Probe illumination volume was minimized using total internal reflection fluorescence microscopy and PAGFP was sparsely photoactivated such that polarized fluorescence identified single probe orientation. Several physiological states of the muscle fiber were characterized revealing two distinct orientation populations in all states called straight and bent conformations. Conformation occupancy probability varies among fiber states with rigor and isometric contraction at extremes where straight or bent conformations predominate, respectively. Comparison to previous work on single rigor cross-bridges at the A-band periphery where myosin concentration is low suggests molecular crowding in the Aband promotes occupancy of the straight myosin conformation (Burghardt et al., 2007, Biophys. J. 93, 2226. The latter may have a role in contraction because it provides additional free energy favoring completion of the cross-bridge power stroke. KeywordsPhotoactivatable green fluorescent protein; photoselected activation; myosin regulatory light chain; single molecule biophysics; molecular crowding; myosin lever arm orientation; human cardiac myosin regulatory light chain Myosin 2 is the actin-dependent molecular motor driving sarcomeric shorting and muscle contraction by transducing chemical free energy from MgATP hydrolysis into directed movement (1,2). The MgATP hydrolysis is coupled to a series of conformational changes in myosin, resulting in a cycle of attachment to the actin filament, strain development, release, and re-attachment. Myosin structural intermediates from the cycle summarized in Scheme 1 † This work was supported by NIH-NIAMS grant R01AR049277 and the Mayo Foundation. *Corresponding author: Telephone: 507 284 8120, Fax 507 284 9349, email: E-mail: burghardt@mayo.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 February 3. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptwere uniquely identified by near ultra-violet circular dichroism spectra and nucleotide sensitive tryptophan fluorescence intensity changes (3)....

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Effect of Metal Cations on the Conformation of Myosin Subfragment-1-ADP-Phosphate Analog Complexes: A Near-UV Circular Dichroism Study

Cited by 21 publications

References 27 publications

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Single Myosin Lever Arm Orientation in a Muscle Fiber Detected with Photoactivatable GFP

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