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.
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Dr Ewa Skrzypczak-Jankun Associate Professor
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-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Mischa Machius
Sent: Thursday, June 12, 2008 10:15 AM
To: [log in to unmask]
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
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Tel: +1 214 645 6381
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