Explanation of a tertiary structure
Shaun D. Black
SHAUN at JASON.UTHCT.EDU
Wed Sep 27 14:05:20 EST 1995
Elias Klein wrote:
>I enjoyed your explanation of forces leading to protein folding. Could
>you expand on this topic a little further to explain the effects of (a)
>ionic strength/pH; (b) chaotropes; (c) surfactants; (d) solvent/water
>mixtures. All of these become important in chromatographic separations.
>Thanks for your comments.
Hi Elias and colleagues,
Glad to expand a bit for you. (a) Effect of Ionic Strength / pH:
Ionic strength really doesn't have a huge effect around the physiological
range. Some proteins will precipitate out of solution if the ionic strength
is very high (e.g. 1.5M ammonium sulfate) or very low (water). Molecularly,
these effects on protein 3D structure are not huge (at least as far as
NMR vs X-ray crystal structures go). Minor effects are due to addition,
substitution, or removal of ions, often cations to the protein. Sorry
for not being more specific but so many examples of different behavior are
out there that this is the best I can do in brief. However, the story is
much different for pH. Physiological pH ranges from about 1 to about 10,
and over this range, huge changes in protein tertiary structure occur. The
primary effect is due to protonation and deprotonation of acidic/basic
side chains and the N- and C-termini that occur at various pHs. The key
side chains are His, Lys, Asp, Glu, Cys, Tyr, and to a lesser extent Arg.
For example as His goes from pH 5 to 7 it is deprotonated and goes from
cationic to neutral (though polar); as the pH of Asp is changed from 3 to 5
it is protonated and goes from neutral (though polar) to anionic. Con-
sequently, the surface charge changes, and with it charge-charge repulsion
and bonding. At low pH (as in the stomach or in a lysosome) the surface
charge is mostly positive, whereas at higher pH the surface is predominantly
anionic. Like charges repel, and local occurrences of this phenonminon can
destabilize local bonding interactions (hydrogen bonds, etc.) that minimally
will change the conformation and maximally will lead to unfolding.
(b) Effect of Chaotropes: Chaotropes such as urea or guanidine are aggressive
hydrogen bonders (donors and acceptors). Thus, they can bind to the surface
of a folded protein and compete for native hydrogen bonds, essential in the
stability of alpha helices, beta sheets, and beta turns. Higher concentrations
compete more effectively and can lead to unfolding.
(c) Effect of Surfactants (Detergents): As amphiphiles, detergents contain
a polar structure where one region is polar and the other is non-polar or
hydrophobic. The principal effect is for the hydrophobic region to "partition"
into non-polar cores in the folded protein. In some cases, such as with
SDS, unfolding occurs readily, either by disruption of hydrophobic cores or
through charge-charge repulsion of adjacently bound detergents (if it is an
ionic detergent). This makes it pretty easy to see why nonionic detergents
and highly bulky detergents (e.g. cholate) have little tendency to denature
(d) Effect of Solvent/Water Mixtures: Solvent/water mixtures (e.g. CH3CN:
water) result in a medium that is lower in dielectric than pure water. In
other words the medium is less polar or more hydrophobic. The extreme case
where the medium is very non-polar causes protein unfolding and "inversion"
by a simple partition or "like dissolves like" effect. Hydrophobic residues
fold to the surface and polar residues form interior cores. At intermediate
ratios, various fractions of native and inverted structure can exist. With
respect to your interest in chromatography, we have found that more-native
proteins chromatograph better on RP-HPLC than do more-denatured ones. In
other words, in good reversed-phase separations of proteins the native
tertiary structure is still somewhat intact during chromatography.
Well, that's all for now. Have fun, and I hope this gets you a bit closer...
Best regards, Shaun
= Shaun D. Black, PhD | Internet address: shaun at jason.uthct.edu =
= Dept. of Biochemistry | University of Texas Health Center, at Tyler =
= World Wide Web: http://pegasus.uthct.edu/UTHCT-Home/Welcome.html =
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