Analysis of the effect of local interactions on protein stability.
BACKGROUND
Protein stability appears to be governed by non-covalent interactions. These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions). The specific role of local interactions is controversial. Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins. However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability. Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability.


RESULTS
To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity. alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing. The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides. Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ([urea]1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m). ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f).


CONCLUSIONS
These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state. Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects. Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions.
Muñoz
V
V
EMBL, Heidelberg, Germany. MUNOZ@embl-heidelberg.de
Cronet
P
P
López-Hernández
E
E
Serrano
L
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D014508:Urea