QUESTIONS: alpha-helix "signals" in proteins

Ben Davis bjd12 at cus.cam.ac.uk
Wed Jul 6 11:04:26 EST 1994


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."



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