| Quick navigation: | Home | Site Map || References | Biography || Copyright | Other copyright | Contact us | Advert | | |
Re: [ccp4bb] Puzzling protein-protein interaction |
||
- Protein crystallographyMain steps:- Protein purification- Crystallisation Special:- Programs for crystallography- X-ray detectors Basic tutorials:- Chemistry- Protein - Peptide - Amino Acids Xtal community:- CCP4BB |
CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: Puzzling protein-protein interaction From: Brett Collins b {- dot -} collins {- at -} IMB {- dot -} UQ {- dot -} EDU {- dot -} AU Date: 2008-09-03 Dear Artem, Sorry I didn't want to scare people with too may details. The pulldowns and ITC were done with the same protein samples expressed in bacteria and purified by the usual ways. For both mutant and wildtype the stoichiometry from ITC was approximately 1:1 (from about 0.85 to about 1.15 range). The ITC experiment was performed over a period of an hour using 18 injections (i.e about 180 second delay between injections and the signal clearly reached baseline again between injections). The pulldowns were perfomed over a similar time period using several washes of His tagged protein + ligand protein bound to beads. Perhaps the only clear difference is that the pulldowns were done in 20 mM Hepes pH 7.5 while the ITC was done in 20 mM Tris pH 8.0 (both with 150 mM NaCl, 1mM DTT). I imagine that the pH may make a difference - but I am not convinced by this explanation. I would still expect to see qualitatively the same result (reduction in binding) at slightly higher pH. Thanks, brett On 03/09/2008, at 7:00 AM, artem@xtals.org wrote: > Dear Brett, > > What was the value of N in your experiments? Did you get a clear "1" > (well, typically 0.8-0.9 is good enough)? Were there differences in N > between the two experiments? (Probably not but I'd rather ask). > > What was peak integration time for the ITC experiment, and how does it > compare with incubation time used in pull-downs? > > How exactly were the pulldowns set up? Did you use pure protein or > was it > an extract of some sort? > > Cheers! > > Artem > >> Dear CCP4 Community, >> >> My apologies for the non-crystallography biochemical >> question but it occurred to me that there are many people >> on this list who are also very good biochemists. >> >> We have just performed an ITC experiment with two proteins >> and measured a Kd of 150 nM, deltaH of -15 kCal/mol, >> deltaS of -15 Cal/mol/K and deltaCp of -2000 J/Mol/K. >> >> We also measured the binding of a mutant of one of these >> proteins predicted from crystal structure to inhibit >> binding of a small fragment of peptide (this is predicted >> to reduce binding slightly but not to affect total binding >> as there is still a large interaction interface that is >> left intact). >> >> This mutant has a Kd of 150 nM as well, but deltaH is -10 >> kCal/mol, delta S is essentially zero, and deltaCp reduces >> in magnitude to about -1500 J/Mol/K as we would predict >> from the change of buried surface area. The ITC data looks >> good and we have repeated the experiments a number of >> times so they are statistically significant. The >> experiments were performed within reasonable concentration >> limits (~10uM protein in the cell so the C-value is about >> 50-100) >> >> Now the puzzle is that the mutant binds less strongly in >> pulldowns (about 50% reduction after several washes) but >> we see an almost identical Kd by ITC despite major changes >> in enthalpy/entropy contributions to binding. The mutant >> and wildtype appear to have identical fold by CD but of >> course there may be small differences. Everything makes >> sense except the lack of Kd change by ITC. >> >> Does anyone have any experience of similar results, or >> more importantly have a possible explanation for them? >> >> Any thoughts greatly appreciated. >> >> Brett Collins >> > ################################ Brett Collins, PhD Group leader Institute for Molecular Bioscience Level 3 North Queensland Bioscience Precinct The University of Queensland St. Lucia, 4072, Qld, AUSTRALIA e-mail: b.collins@imb.uq.edu.au phone: 61-7-3346-2043 FAX: 61-7-3346-2101 website: http://www.imb.uq.edu.au/index.html?page=82433 Courier address: Institute for Molecular Bioscience Queensland Bioscience Precinct Building 80, Services Road University of Queensland St. Lucia, Brisbane Queensland, Australia 4072 ################################ CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
|
| ProteinCrystallography.org: Copyright 2006-2010 by Quid United Ltd |