QUESTIONS: alpha-helix "signals" in proteins

Kenneth Prehoda kenp at tuli
Thu Jul 7 20:16:15 EST 1994


Simon Brocklehurst (Bioc) (smb18 at mole.bio.cam.ac.uk) wrote:
: kenp at banyo (Kenneth Prehoda) writes:

: >:     They're kinetically inaccessible.  That is, if you don't have 
: >: transition states of sufficiently low energy to allow the protein to
: >: pass through them, then you won't get to a particular energy minimum 
: >: (native state).

: >That's my point - they're kinetically inaccessible (in most cases) if
: >they are there at all.  So of what importance are they?

:   Sorry, we're agreed on this point!

:   But I think the pathway from the unfolded to the native state involves 
:   going over energy maxima, and I think this must be where our difference
:   opinion is (right or not?).

:    Maybe now's the time to clarify a few points?

:     1) Do you believe that when proteins fold they, do so along
:        particular pathways that are directed by the sequence?

:     2) Do you think that these pathways involve traversing energy
:        maxima (transition states)?

:     3) If yes to the transition state idea, do you think that these
:        states have some native like structure (e.g. partially native
:        secondary structure)?

:     4) Do you think that rather than going through transition states,
:        there is a roughly downhill path from the unfolded state to the
:        native state i.e. no significant maxima?

:      My answers are:

:        1) Yes

:        2) Yes
:  
:        3) Yes

:        4) No


:      It seems to me that the yes answer to question 3) is particulary
: important to arguments about kinetic control?


: (stuff deleted)

: (talking about side-chain conformations in helices...)
: >Well, the researchers in the field that I have talked to believe
: >that these restrictions do cause energetic differences which is
: >one contribution the the "propensity."

:    I don't disagree that this is important. It's just that you seemed 
:    to imply that alanine residues are helix forming 'cos they don't make 
:    any unfavourable steric nteractions.

:    I was just pointing out that some people think otherwise i.e. they
:    think that alanines intrinsically (i.e. on their own!) like to 
:    be in a helical conformation.  (Personally, I don't have strong opinions 
:    on this matter).

:  (stuff deleted)

: >I wish.  See Gellman et al. for extensive discussions.  My point
: >is that the role of hydrogen bonds in protein stability is
: >entirely _uncertain_ in contrast to your strong opinion otherwise.

:   I never meant to imply that hydrogen bonds were not important
:   contributors to the stability of the native state.

:   I do believe that the hydrophobic side-chains of residues get
:   together at least on the same time-scale as H-bonds form, and 
:   quite likely before.  Thus I don't see how H-bond formation directs
:   folding.

: >For one thing, how do you measure the strength of a hydrogen bond?
:  
:   Tricky do this well:

:   You can do protein engineering experiments to _try_ to estimate
:   the strength of hydrogen bonds involving side-chains (see some of
:   Fersht's work).

:   You can monitor rates of hydron exhange of amide protons by solution homo
:   and heteronuclear magnetic resonance spectroscopy.  But the 
:   interpretation is complicated by motions other than transient
:   breaking of hydrogen bonds.  This does give a handle on main-chain
:   H-bonds though.

:   I'm sure there are loads of ways of doing this, but it's late and
:   I can't think of any off hand!

:   Anyway, depending on the conditions the protein is under, the relative
:   strengths of all kinds of non-covalent interactions will change.

:   I don't want to flog a dead horse, but I think the important question
:   about protein folding is:

:          How does the sequence of the protein limit the number of 
:    conformations that need to be explored to find the native state?

:         My feeling is that this search involves going uphill as well 
:    as downhill on the free energy surface, traversing highly populated 
:    intermediates and less highly populated transitions states.  
:    Characterising the structures of partly folded states (and even
:    completely "unfolded" [whatever that means] states) is obviously an
:    important step forward to understanding protein folding.

:     Do you really not think that understanding the mechanism(s) by
:     which proteins fold is interesting/important?

:   _________________________________________________________________________
:   |
:   |  ,_ o     Simon M. Brocklehurst,
:   | /  //\,   Oxford Centre for Molecular Sciences,
:   |   \>> |   Department of Biochemistry, University of Oxford,
:   |    \\,    Oxford, UK.
:   |           E-mail: smb at bioch.ox.ac.uk
:   |________________________________________________________________________



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