A MYOGLOBIN VARIANT WITH A POLAR SUBSTITUTION IN A CONSERVED HYDROPHOBIC CLUSTER IN THE HEME-BINDING POCKET

Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobi c packing interface between helices G and H in the proximal heme pocke t of horse heart myoglobin, the highly conserved amino acid, Leu104, w as substituted with asparagine, a polar amino acid of similar size. Th e Leu104Asn mutant protein and its recombinant wild-type horse heart m yoglobin counterpart were expressed from synthetic genes in Escherichi a coli. Thermal denaturation of these two recombinant myoglobins, as s tudied by measurement of circular dichroism ellipticity at 222 nm, rev ealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 /- 1 degrees C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degrees C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturb ations, X-ray diffraction techniques were used to determine the three- dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. St ructural comparison of the natural and recombinant wild-type myoglobin s, together with absorption spectroscopic and electron paramagnetic re sonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreas ed thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. E PR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins, Thus, the introduct ion of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significa nt decrease in the t(m) of horse heart myoglobin. (C) 1997 Elsevier Sc ience B.V.