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Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1
Y M Peyser1, A Muhlrad, M M Werber
1Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.
Abstract:
Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122-204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.
Insights
Researchers identified the most reactive tryptophan in rabbit skeletal muscle myosin subfragment-1 (S-1). Using antibody probes, they pinpointed Tryptophan 130 (Trp130) as the primary site of modification by dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS).
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Skeletal muscle myosin subfragment-1 (S-1) contains five tryptophan residues.
- Understanding the reactivity of specific residues is crucial for protein function studies.
- Chemical modification can alter protein structure and function.
Purpose of the Study:
- To identify the most reactive tryptophan residue in rabbit skeletal muscle myosin subfragment-1 (S-1) upon modification with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS).
- To localize the modification site within the S-1 structure.
Main Methods:
- Chemical modification of S-1 with DHNBS.
- Generation of antibodies against DHNBS-modified bovine serum albumin.
- Immune probing of tryptic fragments of modified S-1.
- Isolation and further cleavage of a 27 kDa fragment.
- Peptide mapping and sequencing.
Main Results:
- DHNBS modified approximately 0.8 tryptophan residues per S-1 molecule.
- Antibodies specifically reacted with a 27 kDa tryptic fragment, indicating the reactive tryptophan's location.
- Further cleavage yielded two peptides, with antibodies binding to the smaller peptide (residues 122-204).
- Tryptophan 130 (Trp130) was identified as the sole tryptophan in this reactive peptide.
Conclusions:
- Tryptophan 130 (Trp130) is the most reactive tryptophan residue in rabbit skeletal muscle myosin subfragment-1 (S-1) towards DHNBS modification.
- This finding provides precise residue-level information for future studies on S-1 structure-function relationships.