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Amino Acids

, Volume 35, Issue 2, pp 309–320 | Cite as

X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry

  • G. J. Sathisha
  • Y. K. Subrahmanya Prakash
  • V. B. Chachadi
  • N. N. Nagaraja
  • S. R. Inamdar
  • D. D. Leonidas
  • H. S. Savithri
  • B. M. Swamy
Article

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 

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • G. J. Sathisha
    • 1
  • Y. K. Subrahmanya Prakash
    • 3
  • V. B. Chachadi
    • 1
  • N. N. Nagaraja
    • 1
  • S. R. Inamdar
    • 1
  • D. D. Leonidas
    • 4
  • H. S. Savithri
    • 2
  • B. M. Swamy
    • 1
  1. 1.Department of BiochemistryKarnatak UniversityDharwadIndia
  2. 2.Department of BiochemistryIndian Institute of ScienceBangaloreIndia
  3. 3.Molecular Biophysics UnitIndian Institute of ScienceBangaloreIndia
  4. 4.Institute of Organic and Pharmaceutical ChemistryThe National Hellenic Research FoundationAthensGreece

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