Abstract
A detailed understanding of biological systems at the molecular level requires characterization of structure and dynamics in atomic detail. NMR spectroscopy is uniquely suited to the determination of high resolution solution structures, and has vast potential as a means for examining molecular dynamics because a wide array of nuclear sites within a molecule can be assayed at a variety of different time scales. However, at present we are in the midst of a search to identify how the structural and dynamic information can be fused to generate a unified and comprehensible view for analyzing biomolecular function.
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References
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Discussion
Yuan Xu -1 have one question about your time average constraints. What is the number of conformations that you collected in your time average constraints?
Walter Chazin - What we did in that case was ten 100-picosecond simulations, each was averaged over the last 80 picoseconds, then the ten averaged structures were superimposed. Xu - Do you assign equal probability to those ten conformers?
Chazin - They all have equal probability. The memory function is set so the restraints are satisfied on average over the memory period as opposed to at any particular instance; it’s in essence a mathematical trick.
Xu - When you do the time averaged constraints, is it also possible to do the same thing using Monte Carlo simulations? Chazin - Yes, I don’t see why not.
Brian Sykes -1 am interested in the liganding sidechains in the apo structure. You gave the impression or showed that they really did not move very much. But with all those negative charged sidechains in the loops, wouldn’t they face away from each other in the apo state? Correspondingly, you would expect a real increase in flexibility in the apo structure. Chazin - That’s a multi-tiered question. The first point is that in calbindin D9K, when you look at one site, you have a different answer than the other site. In our preformed site, if it’s really going to be preformed, things need to be oriented in the proper way. The key issue in the N-terminal site is that it doesn’t ligate in the same way as a typical calcium binding protein in that it utilizes a number of neutral backbone carbonyl atoms as opposed to charged sidechain carboxyl atoms. The other EF hand is a more traditional site, and there we see the larger changes. We also see a change in flexibility, at least on the picosecond to nanosecond timescale, that is much more significant. So, there are two stories, I think that this is very integrally related with the nature of calbindin and the fact that it’s an S-100 protein; it has an unusual and atypical binding loop in the N-terminal EF- hand.
Sykes -1 have one supplementary question. I really have no information but I am just interested in the fact that there can be protons on the carboxylic acids; maybe they have a much higher pKa and there are protons in there stabilizing them much like that of water molecules were stabilizing this morning’s story. Have you done anything with water binding or anything like that?
Chazin - We have looked at the waters of hydration in calbindin in the calcium loaded state only and there we do see the two waters of hydration that are involved in ligation. In the C-terminal archetypal site it’s at residue number 9, which is a water mediated ligand, and that water is there. There’s another water that is seen in the crystal structure of calcium- loaded calbindin D9K in the N-terminal ψ-EF-hand and that water is there also. With regard to evidence for unusual pKa’s, we haven’t done any measurements. Well, actually we’ve done measurements but we have not had a chance to analyze those measurements since that was the work of a high school student intern. The data were collected but not really analyzed.
Mengli Cai -1 would like to ask an experimental detail. When you study the structure of the apoprotein, half-saturated and fully saturated, how do you prepare the half saturated protein?
Chazin - The trick there is that we’re using cadmium instead of calcium. Cadmium it turns out, from an inorganic chemistry point of view, has a propensity to bind more covalently than calcium. So it requires a much more specific geometry for ligating than calcium and it doesn’t fit so well in the rigid N-terminal EF hand. It is not optimized and so the magnitude of affinity, the binding constant, is three orders of magnitude lower for cadmium in the N-terminal site than the C-terminal site. So we have sequential binding. The first equivalent of cadmium goes into the C-terminal EF hand and the other hand remains empty. We have recently determined that cadmium still binds cooperatively though and that’s a very important aspect in studying these half saturated states. I should mention someone’s name, Sara Linse, who has worked a lot in this area and who really has done a wonderful job in looking at these cooperative phenomena by doing binding constant measurements. Cai - All right, Thank you.
Gerhard Wagner - Walter, you had quite some discussion about your rmsd’s regarding the comparison with the relaxation parameters. Now, rmsd depends very much on how you align the structures. How do you align the structures?
Chazin - When we do a fitting like for that diagram, where we compare against the order parameters, we try to be as general as possible. However, we try to use well-defined regions of the protein. In particular in the calbindin case, we have used the four helices and the well-defined sidechains for the fitting. When we try and look at the conformational consequences of binding in calbindin’s case, we have an N-terminal EF hand that does not move much and a C-terminal EF hand which does. So we fit the N-terminal EF hand. We do the best fit there and look at what the change is in the C-terminal EF hand. Wagner - But once you start an alignment, you have to identify a single structure used to align them. You said your structure has the lowest rmsd, or least violation? Chazin - What we would do is take the ensemble, determine the geometric average, best fit the geometric averages and then go back, and best fit the ensembles to the superimposed means. If we take two structures and just compare two structures, we take the structure which is closest to the geometric average but which is still a solution, not a refined average, or the average itself, but the single representative structure.
Wagner - Walter, it is going into much detail, but since your geometric average is again dependent on how you initially align the structures, I just want to mention an alternate approach. We have once used a method which is independent of any particular structure. So we have used a bunch of structures (Tim Havel’s idea), taken the distances for each of the structures, and then calculated the averages. It is similar to what Gordon Crippen did when he compared the structures.
Chazin - Right.
Wagner - Take the average distances, use them for distance geometry calculations and you get a cannonical structure which is very much distorted or can be distorted. It is not a realistic structure, but is an independent way of getting alignment completely independent of structure. So this can be done.
Chazin - Yes, I agree most whole heartedly. It is like the distance difference matrix method. I think this is a very important thing. Right now we do ourselves a bit of disservice by relying so much on the rmsd, until some of the other less fitting specific methods do become more common. They also have to be accessible and useable so to speak. I think the rmsd is only valuable for internal comparisons, as things change during the course of refinement or something like that, but otherwise they actually can be dangerous.
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Chazin, W.J. (1996). Incorporating Motional Properties into the Interpretation of Three-dimensional Solution Structures. In: Rao, B.D.N., Kemple, M.D. (eds) NMR as a Structural Tool for Macromolecules. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-0387-9_6
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DOI: https://doi.org/10.1007/978-1-4613-0387-9_6
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