Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state

Molten globule states are partially folded states of proteins which are com pact and contain a high degree of secondary structure but which lack many o f the fixed tertiary interactions associated with the native state. A set o f peptides has been prepared in order to probe the role of local interactio ns in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been ana lyzed using nuclear magnetic resonance, circular dichroism, fluorescence sp ectroscopy and sedimentation equilibrium experiments. A peptide correspondi ng to the first helical region in the native protein, residues 1-13, is onl y slightly helical in isolation, Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Cr osslinking the N- and C-terminal peptides by the native disulfide bond resu lts in almost no increase in structure and there is no evidence for any sig nificant cooperative structure formation over the range of pH 2.2-11.7. The se results demonstrate that there is very little enhancement of local struc ture due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(1 11) disulfide, (C) 2000 Elsevier Science B.V. All rights reserved.