QUESTIONS: alpha-helix "signals" in proteins

SARIDAKIS, VIVIAN C. vivian at vax2.concordia.ca
Fri Jul 15 02:29:00 EST 1994

In article <2vekma$c3i at lyra.csx.cam.ac.uk>, bjd12 at cus.cam.ac.uk (Ben Davis) writes...
>Ken Prehoda (kenp at nmrfam.wisc.edu) wrote:
>: In article <2vdk7s$ss at lyra.csx.cam.ac.uk>
>: Ben Davis, bjd12 at cus.cam.ac.uk writes:
>: >	GIven the amount of work it takes to characterise a denatured state
>: >fully (I'm primarily talking full NMR assignment + std biochemical
>: evidence)
>: >I'm absolutely sure an example of "totally dentaured protein" would get
>: >published, espc. since there aren't any yet ...
>: How can you get a full NMR assignment of a fully denatured protein?
>: There have been many attempts to study denatured proteins by NMR.
>: The problem is there is no dispersion (in sharp contrast to the folded
>: state)
>: in the signals making it impossible to assign them (indicating
>: that there is little, if any structure).  One would expect that if
>: there were local interactions in the unfolded state, they would
>: cause dispersion in the chemical shift.
>	OK. You can get an assignment of proteins under denaturing conditions -
>infact, its what I've spent the last year or so doing.
>	Two main ways of doing it:
>	(1) Use magnetisation transfer from folded ---> unfolded resonances - ie
>get a mixture of folded and unfolded protein (1:1), get correlations between
>the same atom in each state (this is Neri & Wuthrich, 1992, Science
>257,1559-1563, also later again in JMB). This is a tricky way to do it - lot
>of aggregation problems, very crowded spectra, but you can do it.
>	(2) use 13C/15N labelled protein, and triple resonance expts - done
>recently for FK506 Binding Protein (Logan et al, 1994, JMB, 236, 637-648).
>Much better way to do it IMHO - use the dispersion in all 3 dimensions. You
>can also do quite a lot with just 15N labelled protein if the protein is
>small enough.
>	What people tend to see is that, for most (say > 80%) of the protein,
>the assignments are close to random coil values (remember 15N shift is more
>sequence dependant that 1H, so you keep most of this dispersion - very
>useful), but some resonances deviate from random coil, usually by small
>amounts (say approx 0.2ppm max for non-labile protons, 0.4ppm for labile).
>Tie this in with NOE data, you get evidence for non-random coil structures
>being adopted under "denaturing" conditions.
>: Maybe it would help if you would explain what would satisfy a "totally
>: denatured protein?"
>	Very good question - can you ever get a totally random coil protein ? I
>suspect under say 6M GuHCl you'd be looking at "totally" denatured, since
>all the aa would be interacting with solvent rather than other aa
>(hopefully). As it is, proteins denatured by acid, by urea, by heat, by
>truncation seem to show residual or marginal elements of structure.
>	My earlier point was that it'd be good to get an example of a protein
>that *was* totally denatured - just as we need more folded structures, so we
>need more examples of how proteins behace under denaturing conditions.
>: kenp at nmrfam.wisc.edu
>Ben Davis,
>MRC Protein Function and Design,
>Cambridge, UK
>"They can make me do it, but they can't make me do it with dignity."

More information about the Proteins mailing list

Send comments to us at biosci-help [At] net.bio.net