Sathisha, GJ and Prakash, Subrahmanya YK and Chachadi, VB and Nagaraja, NN and Inamdar, SR and Leonidas, D and Savithri, HS and Swamy, BM (2008) X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry. In: Amino Acids, 35 (2). pp. 309-320.
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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 $1.1 \AA$ 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.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Springer.|
|Keywords:||Sclerotium rolfsii lectin;Amino acid sequence;Mass spectrometry;Protein crystal structure.|
|Department/Centre:||Division of Biological Sciences > Molecular Biophysics Unit
Division of Biological Sciences > Biochemistry
|Date Deposited:||16 Oct 2008 06:00|
|Last Modified:||19 Sep 2010 04:50|
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