You may use fluorescence spectroscopy, i.e. to record emission spectra of the protein after 290 nm excitation

Hi there, if there is changes in the chirality of the residues you could hit that in far- or near-UV CD. You don't need big changes in secondary structure to see changes in the spectrum...but of course you need to make the proper control of the buffer and get a high quality spectrum. You can check the papers of Greenfield about this

http://www.nature.com/nprot/journal/v1/n6/abs/nprot.2006.202.html

Near-UV CD and intrinsic fluorescence can be sensitive to tertiary structure changes and are easy to do. If you can make 15N,2H-labeled protein, 2-D NMR could be very informative. FTIR and MS with H-D exchange can also be used to detect conformational changes.

Dear Chetan,

As per your mentioned conditions, along with Near UV-CD spectroscopy, you should perform Fluorescence Spectroscopy of the protein after 290 nm excitation. If your protein have the Tryptophan residue near the region (Preferably 10-20 Angstrom) which you have mentioned, Then you can perform the Fluorescence Spectroscopy after 280 nm excitation. This data can compliment your Near UV-CD Spectroscopy data.

For more info you can reffer this paper

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1301402/pdf/11325713.pdf   

To do this first of all you have to perform these experiment in normal buffer conditions and then you have to perform this by changing the buffer and temperature by performing the thermal denaturation studies using both CD as well as flourascence spectroscopy.

If you don't have any Tryptophan residues in the vicinity or if your protein does not have any Tryptophan residues in your protein, then you can select and mutate an amino acid to Tryptophan (The selected residue should be similar in nature to Trp) in that protein and then perform the above mentioned studies. Make sure your mutation does not alter the proteins function or it should not make the protein an inactive one. for this kind of studies you can reffer this paper.

 http://www.fasebj.org/content/27/8/3054

The problem with near UV CD is that it can tell you such a change has occurred but it cannot reliably tell you what that  the change is. This may or may not be sufficient for your purposes. Tryptophan fluorescence is limited to one site and the effect of mutating to Trp (and mutating all other Trps if necessary) is likely to be larger than the effect of the solvent. The chemical shift is likely to change in different solvents, so the interpretation of HSQC spectra may be difficult.

The best answer is probably hydrogen/deuterium exchange followed by mass spectrometry. You will have to switch back to a buffer free of salt after labeling. The technique is sensitive to hydrogen bonding and solvent accessibility and can (supposedly, I haven't seen a larger benchmark) be calculated from MD trajectories.

If your change can't be resolved by H-D exchange, pretty much the only technique left is comparing the NMR NOESY spectra, unless the conformational change involves a relatively large change in distance for at least one residue pair. In that case, EPR or FRET may be useful.

http://www.sciencedirect.com/science/article/pii/S0969212605003916

None ofthe preceeding answesr are really the solutionto that question.

Modifications of the tuirns and loops (as well as corresponding modifications in helices and sheats) are relevant to secondary structure modifications.

The tailored solution is then  to use VIBRATIONAL-CD SPECTROCOPY which follows the changes ineach part of the secondary structures.

I agree with Jeffrey. The dichroism in the near UV and fluorescence could not say anything interesting. Anyway you said you did tests in silico. You saw conformational changes, but I hope that they are derived from a simulation using molecular dynamics in the presence of explicit water at controlled pH, otherwise I have strong doubts that what you saw can be trusted. Even in my opinion, hydrogen / deuterium exchange followed by mass spectrometry is one of the best option.

There is a lot of good advice above, and also direct, simple studies on the CD  and ORD studies on bends and loop-like structures go back a long way (e.g. “Circular dichroism of beta turns in peptides and proteins”, C. Allen Bush , Susanta K. Sarkar , Kenneth D. Kopple, Biochemistry, 1978, 17 (23), pp 4951–4954). To some extent it is true that “The dichroism in the near UV and fluorescence could not say anything interesting.”, at least one cannot easily say anything cheaply and not in absolute rather than relative terms.   The problem really is: what exactly do you mean by conformational changes, what exactly do you mean by protein structure, and (not least) what exactly do you actually mean by turn and loop?  With emphasis the word “changes” here, however, it is of course typically easier to detect change both in a static sense of differences (equilibrium studies) and particularly a dynamic one (kinetic studies), which also goes back a long way: e.g., our “The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation”, B Robson and R H Pain, Biochem. J. (1976) 155 (331–344) To try and make sense of it at the level kind of atomic detail that you  believe you are seeing as indicative of reality  in silico,  however, really requires a lot of calibration, meaning not only perturbation by solvent but also genetic engineering  or chemical modification studies for the specific region of peptide or protein of interest, especially where loops are concerned.  Even today, I think that’s (at very least) a Master’s thesis project than a high throughput routine test.

Near UV circular dichroism investigate tertiary protein structure via absorption of left handed and right handed polarized light