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Electrostatic interactions in wild-type and mutant recombinant human myoglobins
R Varadarajan1, D G Lambright, S G Boxer
1Department of Chemistry, Stanford University, California 94305.
Biochemistry
|May 2, 1989
Summary
Human myoglobin (Mb) mutants tolerated substitutions at Val68, revealing insights into protein interior polarity. Charge stabilization depended on heme iron interactions, affecting proton uptake during reduction and cyanide binding.
Area of Science:
- Biochemistry
- Protein Engineering
- Spectroscopy
Background:
- Myoglobin (Mb) is a crucial protein for oxygen transport.
- Understanding the role of buried residues is key to protein function.
- Site-directed mutagenesis allows investigation of specific amino acid substitutions.
Purpose of the Study:
- To investigate the effects of substituting the buried hydrophobic residue Val68 in human myoglobin with Asn, Asp, and Glu.
- To characterize the biophysical properties of these Mb mutants.
- To explore the stabilization of charge within the protein interior and its impact on heme interactions and chlorophyll derivative spectra.
Main Methods:
- Site-directed mutagenesis to create Val68 mutants (Asn, Asp, Glu).
- Protein purification and characterization using isoelectric focusing, absorption, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy.
- Reconstitution of apoproteins with zinc pyrochlorophyllide a.
- Spectroscopic analysis of wild-type and mutant proteins.
Main Results:
- Human myoglobin tolerates substitution of Val68 with Asn, Asp, and Glu.
- Negative charge at residue 68 is stabilized by Coulombic interaction with heme iron in Asp/Glu mutants.
- Proton uptake accompanies reduction and cyanide binding in Asp/Glu mutants due to charge stabilization.
- Spectra of reconstituted chlorophyll derivatives were similar across wild-type and mutant proteins.
- Apparent pKa of buried glutamate in the Glu mutant was 8.9, indicating a nonpolar protein interior.
Conclusions:
- The protein interior's polarity significantly influences the stabilization of buried charges.
- Myoglobin's structure can accommodate significant changes at buried positions without compromising overall stability.
- The findings challenge the simple point charge model for chlorophyll spectra in photosynthetic proteins.
- The study provides a quantitative measure of the protein interior's nonpolar nature.