Protein folding: assignment of the energetic changes of reversible chemical modifications to the folded or unfolded states

Citations Over TimeTop 17% of 1992 papers

Abstract

Reversible chemical modifications of a series of single cysteine-containing variants of T4 lysozyme combined with thermal denaturation studies have been used to study the effects of these modifications on the stability of the protein. This allows dissection of the energetic effects of the modification on both the native and denatured states of this protein. At some sites modifications with various chemical reagents have essentially no effect on the stability of the protein, while at others, substantial changes in stability are observed. For example, chemical modification of cysteine at site 146 by cystamine (+NH3CH2CH2SSCH2-CH2NH3+) to form the mixed disulfide lowers the stability of the protein by about 1.1 kcal/mol. The reduction in the free energy of folding caused by the chemical modification is attributed to the destabilization of native state (0.9 kcal/mol), with only a relatively small effect from stabilization of the denatured state (0.2 kcal/mol). Chemical modifications of T4 lysozyme at site 146 with various chemical reagents show that the stability of the protein is lowered by a positively charged group and is relatively independent of the size of the side chains. This approach allows the investigation of the thermodynamic consequences of the reversible insertion of a wide variety of chemical entities at specific sites in proteins and, most importantly, allows dissection of the contribution of the chemical modifications to both the folded and unfolding states. It can be applied to almost any suitable macromolecular system.

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