X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry Article First Online: 28 December 2007 Received: 11 September 2007 Accepted: 16 October 2007 DOI:
Cite this article as: Sathisha, G., Subrahmanya Prakash, Y., Chachadi, V. et al. Amino Acids (2008) 35: 309. doi:10.1007/s00726-007-0624-y Summary.
X-ray crystallography, although a powerful technique for determining the three-dimensional structure of proteins, poses inherent problems in assigning the primary structure in residues Asp/Asn and Glu/Gln since these cannot be distinguished decisively in the electron density maps. In our recently published X-ray crystal structure of the
Sclerotium rolfsii lectin (SRL) at 1.1 Å resolution, amino acid sequence was initially deduced from the electron density map and residues Asp/Asn and Glu/Gln were assigned by considering their hydrogen bonding potential within their structural neighborhood. Attempts to verify the sequence by Edman sequencing were not successful as the N terminus of the protein was blocked. Mass spectrometry was applied to verify and resolve the ambiguities in the SRL X-ray crystal structure deduced sequence. From the Matrix assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI TOF-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis of tryptic and chymotryptic peptides of SRL, we could confirm and correct the sequence at five locations with respect to Asp/Asn and Glu/Gln. Analysis data also confirmed the positions of Leu/Ile, Gln/Lys residues and the sequence covering 118 of the total 141 residues accounting to 83.68% of the earlier deduced sequence of SRL. Keywords: Sclerotium rolfsii lectin – Amino acid sequence – Mass spectrometry – Protein crystal structure
Authors’ address: Dr. B. M. Swamy, Department of Biochemistry, Karnatak University, Dharwad-580 003, Karnataka, India
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