High-throughput limited proteolysis/mass spectrometry for protein domain elucidation.
High-resolution structural information is important for improving our understanding of protein function in vitro and in vivo and providing information to enable drug discovery. The process leading to X-ray structure determination is often time consuming and labor intensive. It requires informed decisions in expression construct design, expression host selection, and strategies for protein purification, crystallization and structure determination. Previously published studies have demonstrated that compact globular domains defined by limited proteolysis represent good candidates for production of diffraction quality crystals [1-7]. Integration of mass spectrometry and proteolysis experiments can provide accurate definition of domain boundaries at unprecedented rates. We have conducted a critical evaluation of this approach with 400 target proteins produced by SGX (Structural GenomiX, Inc.) for the New York Structural GenomiX Research Consortium (NYSGXRC; http://www.nysgxrc.org) under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative (http://www.nigms.nih.gov/psi). The objectives of this study were to develop parallel/automated protocols for proteolytic digestion and data acquisition for multiple proteins, and to carry out a systematic study to correlate domain definition via proteolysis with outcomes of crystallization and structure determination attempts. Initial results from this work demonstrate that proteins yielding diffraction quality crystals are typically resistant to proteolysis. Large-scale sub cloning and subsequent testing of expression, solubility, and crystallizability of proteolytically defined truncations is currently underway.
Gao
Xia
X
Structural GenomiX, Inc, 10505 Roselle Street, San Diego, CA 92121, USA.
Bain
Kevin
K
Bonanno
Jeffery B
JB
Buchanan
Michelle
M
Henderson
Davin
D
Lorimer
Don
D
Marsh
Curtis
C
Reynes
Julie A
JA
Sauder
J Michael
JM
Schwinn
Ken
K
Thai
Chau
C
Burley
Stephen K
SK
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D011506:Proteins; D010447:Peptide Hydrolases