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

Mühlrád, András; Morales, Manuel F.

doi:10.1073/pnas.81.4.1003

Cited by 48 publications

(18 citation statements)

References 22 publications

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“…The notion was applied years ago to myosin with the discovery of conserved proteolysis products suggesting they constitute functional domains (71,72). Functional domains defined by proteolysis proved to be of limited practical value in myosin but the idea remains inviting for use in new applications in protein engineering.…”

Section: Discussionmentioning

confidence: 99%

An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

Halstead

Ajtai

Penheiter

et al. 2007

Biophysical Journal

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The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network.

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Section: Discussionmentioning

confidence: 99%

An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

Halstead

Ajtai

Penheiter

et al. 2007

Biophysical Journal

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“…Finally, the competition between the 23-kDa fragment and S-l for actin was studied by measuring the effect of the fragment on the actin-activated ATPase of S-l (Muhlrad & Morales, 1984). To a reaction mixture containing S-l and F-actin was added 23-kDa fragment in increasing concentrations, and the ATPase activity was measured (Figure 9).…”

Section: Resultsmentioning

confidence: 99%

“…The N-terminal 23-kDa fragment of the S-l heavy chain was isolated for the first time from the tryptic digest of S-l following dissociation of the heavy-chain fragments in guanidine hydrochloride. A similar procedure was employed for the isolation of the other two heavy-chain fragments (Muhlrad & Morales, 1984; Muhlrad et al, 1986;Ueno et al" 1985).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, the isolation, which is carried out in the presence of denaturants, has to be followed by renaturation, in the absence of denaturants. We recently isolated, renatured, and characterized the structure and function of the 20-and 50-kDa fragments of the S-l heavy chain and found that both fragments contain actin binding sites (Muhlrad & Morales, 1984;Muhlrad et al, 1986). Chaussepied et al (1986b,c) isolated various C-terminal fragments (20, 22, and 30 kDa) of S-l complexed with LC3 alkali light chain and observed that this complex interacts with actin.…”

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confidence: 99%

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Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1

Mühlrád¹

1989

Biochemistry

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The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.

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“…The 20-kDa fragment attached to the myosin rod, with apparent tertiary structure, can be isolated and seen in negatively stained electron micrographs (Winkelmann et al, 1984). The 20-kDa fragment also can be isolated from S1(T) and when freed from denaturant will bind to actin (Muhlrad & Morales, 1984), as will a 10-kDa fragment derived from the 20-kDa fragment (Katoh et al, 1985). Skeletal muscle SI is flexible, suggesting it is segmented (Highsmith & Eden, 1986).…”

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confidence: 99%

Limited trypsinolysis changes the structural dynamics of myosin subfragment-1

Highsmith¹,

Eden²

1987

Biochemistry

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The effects of limited trypsinolysis of myosin subfragment 1 (S1) on its structural dynamics were investigated by using the method of transient electric birefringence. Conversion of S1 by trypsin to produce S1 (T) did not change the specific Kerr constant [(8.1 +/- 0.3) X 10(-7) and (8.0 +/- 0.3) X 10(-7) cm2/statvolt2 for S1(T) and S1, respectively] or the degree of alignment in a weak electric field, suggesting that the size of S1 and its permanent electric dipole moment are not modified by trypsin. On the other hand, the relaxation time for the field-free rotation, after achieving a steady-state birefringence signal, was reduced from 316 ns for S1 to 269 ns for S1(T), at 3.7 degrees C, suggesting that trypsinolysis increases the flexibility of the connections between S1 segments or introduces additional segmental motions. For both S1 and S1(T), the rate of decay for a steady-state signal was independent of the field strength, between 3.34 and 20.3 statvolt/cm. Shortening the duration of the weak electric field pulses to 0.35 microseconds, so that steady-state signals were not achieved, decreased the relaxation times for S1 and S1(T) to 240 and 210 ns, respectively, which is consistent with the segmented flexible S1 structure proposed earlier [Highsmith, S., & Eden, D. (1986) Biochemistry 25, 2237]. When the strength of the electric field was increased to above 10 statvolt/cm, in order to make the interaction energy for the S1(T) electric dipole moment in the electric field greater than the thermal energy, the relaxation time after a 0.35-microseconds pulse decreased from 210 to 170 ns as the field was increased from 7 to 20 statvolt/cm. (ABSTRACT TRUNCATED AT 250 WORDS)

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

Cited by 48 publications

References 22 publications

An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1

Limited trypsinolysis changes the structural dynamics of myosin subfragment-1

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