Fundamental processes of protein folding: measuring the energetic balance between helix formation and hydrophobic interactions.

Theories of protein folding often consider contributions from three fundamental elements: loops, hydrophobic interactions, and secondary structures. The pathway of protein folding, the rate of folding, and the final folded structure should be predictable if the energetic contributions to folding of these fundamental factors were properly understood. alphatalpha is a helix-turn-helix peptide that was developed by de novo design to provide a model system for the study of these important elements of protein folding. Hydrogen exchange experiments were performed on selectively 15N-labeled alphatalpha and used to calculate the stability of hydrogen bonds within the peptide. The resulting pattern of hydrogen bond stability was analyzed using a version of Lifson-Roig model that was extended to include a statistical parameter for tertiary interactions. This parameter, x, represents the additional statistical weight conferred upon a helical state by a tertiary contact. The hydrogen exchange data is most closely fit by the XHC model with an x parameter of 9.25. Thus the statistical weight of a hydrophobic tertiary contact is approximately 5.8x the statistical weight for helix formation by alanine. The value for the x parameter derived from this study should provide a basis for the understanding of the relationship between hydrophobic cluster formation and secondary structure formation during the early stages of protein folding.

Resistance of antivenom proteins to foaming-induced denaturation.

Reconstitution of lyophilized antivenom is usually accomplished by gentle swirling for sometimes as much as 45min. Gentle resuspension is employed in order to avoid foaming the antivenom, which could have a variety of consequences including unfolding and denaturation of the protein, thus rendering it inactive. However, foaming of antivenom might cause only a small portion of the total protein to unfold or the protein may refold as foaming subsides. In this report, the effects of intentional severe foaming of three antivenom preparations are tested. Samples of gently resuspended antivenoms were subject to severe foaming by mechanical means, then tested for precipitation by examining the amounts of protein remaining in solution, for structural changes by circular dichroism spectroscopy and for activity changes by ELISA. In all cases, either no or minimal changes in antivenom properties were observed. These results indicate that severe intentional foaming does not substantially affect the properties of the antivenoms. It may be possible to employ more vigorous resuspension methods without affecting the efficacy of the antivenom. This could lead to a significant decrease in the time between envenomation and administration of antivenom.

Understanding protein folding through peptide models.

From the time it was recognized that proteins are made up primarily of secondary structures, theories of protein folding have used secondary structural elements as important building blocks. Peptides have played a central role in elucidating the factors that stabilize individual elements of secondary structure and are now being employed to study higher levels of organization. The control of conformation in peptides has taken on new relevance with the realization that protein folding plays a central role in many disease states.