X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry.
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 A 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.
Sathisha
G J
GJ
Department of Biochemistry, Karnatak University, Dharwad, India.
Prakash
Y K Subrahmanya
YK
Chachadi
V B
VB
Nagaraja
N N
NN
Inamdar
S R
SR
Leonidas
D D
DD
Savithri
H S
HS
Swamy
B M
BM
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods; D013997:Time Factors
D037102:Lectins