Dear Marc,

Energy minimisation is most of the time performed with constrains on all heavy atoms, which means that it will only try to minimise the energy given a specific fold. In other words, minimisation will move the side chains so that there is no violation of van der walls overlaps between atoms (aka clashes). What you did is just to model the wt protein with a mutation and not the mutant itself. To detect changing in folding, you need to perform molecular dynamics using the Gromacs software for instance and possibly with annealing to input some energy to allow the mutated wild type protein to pass energetic barriers that keep the wild type fold as it is even though you introduce a change. It is most likely that changing any amino acid by a proline will have a strong impact on folding. However, only dynamics and not minimisation alone will possibly show any effect of the mutation on the protein folding.

Furthermore, bioinformatics and molecular modelling/dynamics are just tool to guide future experiments and conclusions. I am doing a lot of molecular dynamics simulation but in the end, biological validation (e.g. structure function analysis, transfection...) is the only type of experiment that allow to draw conclusions on the impact of a mutation on a protein, specially if the mutation has never been described before.

Christine Slingsby has since our publication demonstrated the pathogenicity of the mutation and has showed with her colleagues the dramatic effect of the mutation . i do agree with your suggestions but structural methods constitute the best mehods for validating the pathogenicity. The in vivo experiments using the mutant protein are of couse extremely useful and complementary. Nevertheless, ultimately, a only a knock in mouse and or a transgenic model provides the final demonstration in vitro experiments will never replace in vivo demonstration.

The Institutes of Molecular Dynamics in Israel and other countries has provided a lot of results based as you said on computational biology and modeling and experiments.

J Mol Biol. 2004 Oct 15;343(2):435-44.

The P23T cataract mutation causes loss of solubility of folded gammaD-crystallin.

Evans P, Wyatt K, Wistow GJ, Bateman OA, Wallace BA, Slingsby C.

Source

Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK.

Abstract

Mutations in the human gammaD-crystallin gene have been linked to several types of congenital cataracts. In particular, the Pro23 to Thr (P23T) mutation of human gammaD crystallin has been linked to cerulean, lamellar, coralliform, and fasciculiform congenital cataracts. We have expressed and purified wild-type human gammaD, P23T, and the Pro23 to Ser23 (P23S) mutant. Our measurements show that P23T is significantly less soluble than wild-type human gammaD, with P23S having an intermediate solubility. Using synchrotron radiation circular dichroism spectroscopy, we have determined that the P23T mutant has a slightly increased content of beta-sheet, which may be attributed to the extension of an edge beta-strand due to the substitution of Pro23 with a residue able to form hydrogen bonds. Neither of the point mutations appears to have reduced the thermal stability of the protein significantly, nor its resistance to guanidine hydrochloride-induced unfolding. These results suggest that insolubility, rather than loss of stability, is the primary basis for P23T congenital cataracts.

Incidentally is one of your relative a specialist of neonatalogy ????

My dad..

I worked as a medical student and as a young MD at institut de puericulture de Paris. I was fortunate to work in this wonderful institution given by the amercan government to France after the Liberation of Paris. There was no "puericultrice" in France before the arrival of the US army in Paris. I was lucky to work under the supervision of Pr SATGE, Pr CHARLAS whos saved my life from an acute dehydration when i was sixth month old in Morocco, I worked with Dr Voyer and with Dr Miche VODOVAR if my memory is correct. He was already at that time recognized as one of the best neonatalogist and pediatrician of France while he could not get the CES of pediatrics foe obscure reasons or just because he was always saving lives rather than studying the famous "questions" one has to know by heart . I heard that he finally made it and probably was promoted . If he has not been appointed Professor as Voyer was, it is a shame . If he has been , it is OK . We had a wonderful team at Institut de puériculture de Paris with Miss NARBOUTON (MAjor de l'internat des hôpitaux de Paris).....and others without forgeting the PUERICULTRICES able to DRIVE LA VISITE......and to correct the diagnoses and prescriptions of the young and even old residents (Internes) . Whereas I was trained in Ophthalmology, I took long Nights working at the IPP. It is likely that your Dad does not remember me.

Actually he remembers you. I my memory is correct, our path already crossed when I was in PhD when you contacted my then boss, Bruno Lemaitre for some experiments. My dad has now retired and what was the IPP was "bought" by Necker.

I indeed contacted Bruno LEMAITRE and I invited him to give a seminar in NECKER.

The Dean wanted him to establish a full team......

He preferred the CGM and is now in Lausanne.

I think he is genuine about Hoffmann's behaviour . I read his blog....

IPP had a long history of institutional links with Necker Enfants Malades. This is why I worked at IPP as "EXTERNE" in DCEM4 or DCEM2 and latter on for the wonderful atmosphere at IPP