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Well-ordered internal amino acids can contribute significantly to the stability
of proteins. To investigate the importance of the hydrophobic packing interface
between helices G and H in the proximal heme pocket of horse heart myoglobin,
the highly conserved amino acid, Leu104, was substituted with asparagine, a
polar amino acid of similar size. The Leu104Asn mutant protein and its
recombinant wild-type horse heart myoglobin counterpart were expressed from
synthetic genes in Escherichia coli. Thermal denaturation of these two
recombinant myoglobins, as studied by measurement of circular dichroism
ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly
lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin
(81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C
decrease in thermal stability was associated with structural perturbations,
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. Structural comparison of the natural and
recombinant wild-type myoglobins, together with absorption spectroscopic and
electron paramagnetic resonance (EPR) analyses, confirmed the proper expression
and folding of the recombinant protein in E. coli. Surprisingly, despite the
decreased thermal stability of the Leu104Asn mutant, there are no significant
structural differences between the mutant and wild-type myoglobins. EPR and
absorption spectroscopic analyses further confirmed the similar nature of the
heme iron centres in both proteins. Thus, the introduction 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
significant decrease in the t(m) of horse heart myoglobin.
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