Characterization of MB-1 - A dimeric helical protein with a compact core

MB-1 is a de-nova protein designed to incorporate a large number of the nut ritionally important amino acids methionine, lysine, leucine and threonine into a stable four-helix bundle protein. MB-1 has been expressed and purifi ed from Escherichia call, indicating it was resistant to intracellular prot eases [Beauregard, M., Dupont, C., Teather, R.M. & Hefford, M.A. (1995) Bio /Technology 13, 974]. Here we report an analysis of the secondary, tertiary and quaternary structures in MB-1 using circular dichroism, fluorospectros copy and size-exclusion chromatography. Our data indicate that the MB-I str ucture is close to the target structure, an cl-helical bundle, in many resp ects and is highly helical in solution. The single tyrosine incorporated in to the designed protein as a spectrocopic probe of tertiary structure, is b uried in a compact, folded core and becomes accessible on protein denaturat ion, as per design. Furthermore, MB-1 was found to be native-like in many r espects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defi ned; and (c) MB-I has very low affinity for 8-anilino-1-naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles th at of other native proteins. On the other hand, our analysis revealed two a spects that command further attention. The folding stability of MB-1 as ass essed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size-exclusion chromatography e xperiments and analysis of MB-1 denaturation indicate that MB-1 is dimeric, not monomeric as designed. In light of these results, the utility and the current limitations of our design approach are discussed.