Characterization of the tertiary structure of the de novo designed protein MB-1

Milk Bundle-1 (MB-1) is a de novo designed protein with 100 amino acids, having a molecular weight of 11.4 kilodaltons. MB-1 is enriched with 57% of selected essential amino acids (methionine, threonine, lysine and leucine), which are known to be limiting in dairy cattle. Recently, on the basis of a digestibility study, MB-1 was predicted to be unstable in rumen conditions.

Characterization of the protein's structure was achieved using fluorescence spectroscopy (steady state measurements). MB-1 contains one tyrosine at position 62, expected to be in position "d" of helix III, in the hydrophobic core. Data obtained using fluorescence quenching indicates that the tyrosine is protected from the solvent in the putative hydrophobic core, as per design.

Once it was established that MB-1 was not misfolded, further experiments were done to assess the fluidity of its hydrophobic core. For this, the amphiphillic dye ANSA was used. Results obtained for MB-1 compare favourably to those of many natural proteins, suggesting that MB-1 has achieved some degree of nativeness. Interestingly, MB-1 was found to exclude ANSA from its hydrophobic core more efficiently than all other de novo designed proteins reported to date.

Finally, an analysis of folding thermodynamics of MB-1 was attempted. It was found that the fluorescence intensity of tyrosine was not sensitive to unfolding, making thermodynamic data impossible to obtain.

Analysis of the data on MB-1 as compared to other natural proteins indicates that MB-1 is folded and compact. The lack of resistance to proteases must be caused by other factors other than the lack of compactness or misfolding. (Abstract shortened by UMI.).