Characterization of MB-1. A dimeric helical protein with a compact core.
MB-1 is a de-novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four-helix bundle protein. MB-1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases [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, fluorospectroscopy and size-exclusion chromatography. Our data indicate that the MB-1 structure is close to the target structure, an alpha-helical bundle, in many respects and is highly helical in solution. The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design. Furthermore, MB-1 was found to be native-like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB-1 has very low affinity for 8-anilino-1-naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins. On the other hand, our analysis revealed two aspects that command further attention. The folding stability of MB-1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size-exclusion chromatography experiments 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.
Hefford
M A
MA
Center for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Canada. mhefford@emerald.hwc.ca
Dupont
C
C
MacCallum
J
J
Parker
M H
MH
Beauregard
M
M
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004044:Dietary Proteins; D019281:Dimerization; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004044:Dietary Proteins; C107457:MB-1synthetic dietary protein