High-precision, high-throughput stability determinations facilitated by robotics and a semiautomated titrating fluorometer.
The use of statistical modeling to test hypotheses concerning the determinants of protein structure requires stability data (e.g., the free energy of denaturation in H(2)O, DeltaG(HOH)) from hundreds of protein mutants. Fluorescence-monitored chemical denaturation provides a convenient method for high-precision, high-throughput DeltaG(HOH) determination. For eglin c we find that a throughput of about 20 min per protein can be attained in a two-channel semiautomated titrating fluorometer. We find also that the use of robotics for protein purification and preparation of the solutions for chemical denaturation gives highly precise DeltaG(HOH) values in which the standard deviation of values from multiple preparations (+/-0.051 kcal/mol) differs very little from multiple measurements from a single preparation (+/-0.040 kcal/mol). Since the variance introduced into model fitting by DeltaG(HOH) increases as the square of measurement error, there is a premium on precision. In fact, the fraction of stability behavior explicable by otherwise perfect models goes from 98% to only 50% over the error range commonly reported for chemical denaturation measurements (0.1-0.6 kcal/mol). We have found that the precision of chemical denaturation DeltaG(HOH) measurements depends most heavily on the precision of the instrument used, followed by protein purity and the capacity to precisely prepare the solutions used for titrations.
Edgell
Marshall Hall
MH
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599, USA. marshall@med.unc.edu
Sims
Dorothy A
DA
Pielak
Gary J
GJ
Yi
Fang
F
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D019791:Guanidine / Q000737:chemistry; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D012371:Robotics; D015843:Serpins / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; C028656:eglin proteinase inhibitors; D019791:Guanidine