Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination.
The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.
Vedadi
Masoud
M
Structural Genomics Consortium, University of Toronto, 100 College Street, Toronto, ON, Canada M5G 1L5.
Niesen
Frank H
FH
Allali-Hassani
Abdellah
A
Fedorov
Oleg Y
OY
Finerty
Patrick J
PJ
Wasney
Gregory A
GA
Yeung
Ron
R
Arrowsmith
Cheryl
C
Ball
Linda J
LJ
Berglund
Helena
H
Hui
Raymond
R
Marsden
Brian D
BD
Nordlund
Pär
P
Sundstrom
Michael
M
Weigelt
Johan
J
Edwards
Aled M
AM
D000818:Animals; D019295:Computational Biology; D003460:Crystallization; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D008024:Ligands; D011506:Proteins