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 compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions 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 analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only 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. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure 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(111) disulfide.

Effects of sequential proline substitutions on amyloid formation by human amylin20-29.

Amylin, also known as islet amyloid polypeptide (IAPP), is the major protein component of the fibril deposits found in the pancreas of individuals with type II diabetes. The central region of amylin, residues 20-29, has been implicated as a key determinate of amyloid formation. To establish which positions are most important for amyloid formation, the wild-type sequence of the 20-29 fragment and a set of 10 variants have been synthesized in which a proline was placed at each position. Proline is energetically unfavorable in the extended cross-beta structure found in amyloid. If a particular position is critical for amyloid formation, then substitution with a proline should inhibit amyloid formation. A proline substitution at any position inhibited aggregation and amyloid formation. Substitution of Asn22, Gly24, and residues 26-28 had the largest effect. Fourier transform infrared (FTIR) spectroscopy showed little secondary structure in these peptides, and transmission electron microscopy (TEM) showed mostly amorphous material. The peptides were much more soluble than the wild-type sequence, and no birefringence was observed with Congo Red staining. Proline substitutions at the N (residues 20 and 21) and C termini showed the least effect. These peptides showed the classic fibril morphology, a significant amount of beta-sheet structure, and exhibited green birefringence when stained with Congo Red. The results indicate that residues 22, 24, and 26-28 play a key role in formation of amyloid by amylin. Positions 23 and 25 also appear to be important, but may be less critical than positions 22, 24, and 26-28.

A role for the C-terminus of calcitonin in aggregation and gel formation: a comparative study of C-terminal fragments of human and salmon calcitonin.

Calcitonin is used in therapy for osteoporosis and Paget's disease. In vitro, human calcitonin forms thick gels which limits its usefulness as a therapeutic, and consequently salmon calcitonin which is less prone to aggregate is commonly used instead. In order to probe the role of the C-terminal region of the molecule in association and gel formation we have prepared a set of three peptides corresponding to the C-terminal regions of salmon calcitonin, human calcitonin and a mutant of human calcitonin in which Pro-23 is substituted with Ala. The peptides are largely disordered in their monomeric state as judged by CD and FTIR. All three peptides aggregate and form gels. Both human peptides form a gel much faster than the salmon peptide and the proline to alanine mutant forms a gel faster than the wildtype human peptide. Gel formation by all three peptides is slower than for intact human calcitonin. CD indicates a difference in conformation for the human fragment but not for the salmon fragment between the monomeric state and the gel state. FTIR experiments suggest the presence of beta-structure in the gel derived from the human peptide but not in the gel derived from the salmon peptide. These results show that there are clear differences in the association properties of the peptides and point to a potential role for the C-terminal region of calcitonin in controlling aggregation/gel formation.