Let us not forget about the very fact that X-ray is an ionizing radiation and a potent creator of radicals! Please see these references - seeing a crystal that was purple in the middle (0.3mm radius of beam) and perfectly yellow at edges (crystal was >0.5mm long) was an eye opening experience. How many of you keep looking at the crystal during data collection in color? And pay attention to it at all? Not every reaction makes such dramatic effect either - it could as well be 'silent'. Even in this case...I had to put a real fight to publish because one reviewer accused me of some voo-doo (not in such words, but...) saying that I cannot find in the structure what I did not put in. He he he he I wonder how many examples of that are out and documented? :) Ewa Int. J. Molecular Medicine 12(1), 17-24, 2003 and 6, 521-6, 2000.
******************************************************** Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: [EMAIL PROTECTED] Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa ******************************************************** -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mischa Machius Sent: Thursday, June 12, 2008 10:15 AM To: [email protected] Subject: Re: [ccp4bb] Activity of a mutant enzyme compared to wild type - puzzle I assume you are talking about a sugar-binding enzyme ;) I have some aspects to consider in addition to what Artem raises. Many effects of a mutation are not recognizable in a static crystal structure or even in an NMR structure. For example, it is usually difficult to assess the thermodynamics of substrate binding, not to mention the kinetics. Multi-valent substrates usually display some sort of cooperativity for the binding process, which you might have affected by mutating one of the subsites. You might be able to obtain some hints from a Michaelis- Menten analysis of the mutant compared to the wild type, but that would only be a start. Your crystallographic result of a less occupied substrate-binding site for the mutant serves as a hint as well, but such results are hardly conclusive. You will have to follow up with more rigorous methods, such as ITC (thermodynamics of binding) and time-resolved methods (kinetics of binding). One example of an effect of a mutation that is usually not recognizable in a crystal structure has to do with substrate guiding. In this case, the mutation has changed the surface of the protein, thus affecting how well the multi-valent substrate can approach and wiggle itself into the binding site. Once in the binding site, it is structurally virtually indistinguishable from the wild-type. Ah, the nightmares of interpreting crystal structures in terms of biology! Good luck! Best - MM On Jun 11, 2008, at 7:21 PM, Narayanan Ramasubbu wrote: > Dear all: > I have a single residue mutant whose enzyme activity is about 50% of > the wild type. Interestingly, the mutation > is in a region that involves a secondary site but not the active > site. The two structures with or without ligands > fit well (0.18 A) and the metal binding and cofactor binding sites > are all preserved in the mutant. The one difference > noticed is that the ligand does not fill the active site (partially > occupied subsites) unlike the wild type where all the > subsites are occupied. Water structure around the actives site > residues are "identical". > > I looked at the electrostatics and both surfaces look similar (not > an expert). > > There are some residues whose sides chains show some positional > disorder and these residues are at the edges of the > active site. > > The resolution of the both data sets are 1.5A. > The mutant enzyme was derived by MR. > > One another possibility that I want to look at is to compare the > compactness of the two enzyme structures. > What is the best way to compare that? I am wondering whether the > "breathing" that was mentioned for some enzymes > might be playing a role in the mutant enzyme. > > Also, I would appreciate comments on other possible explanations for > this unusual (?) behavior. > > > Thanks a lot > > Subbu -------------------------------------------------------------------------------- Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
