期刊
PROTEIN ENGINEERING DESIGN & SELECTION
卷 18, 期 9, 页码 425-433出版社
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzi051
关键词
alpha-lactalbumin; calcium binding; electrostatic interactions; site-directed mutagenesis; thermal stability
资金
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0802141] Funding Source: National Science Foundation
- NIGMS NIH HHS [GM56970] Funding Source: Medline
It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca2+-loaded alpha-lactalbumin were calculated using a Tanford-Kirkwood algorithm with either solvent accessibility correction or using a finite difference Poisson-Boltzmann method. The analysis revealed two major regions of alpha-lactalbumin that possessed highly unfavorable electrostatic potentials: (a) the Ca2+-binding loop and its neighboring residues and (b) the N-terminal region of the protein. Several individual mutants were prepared to neutralize specific individual surface acidic amino acids at both the N-terminus and Ca2+-binding loop of bovine alpha-lactalbumin. These mutants were characterized by intrinsic fluorescence, differential scanning microcalorimetry and circular dichroism. The structural and thermodynamic data agree in every case with the theoretical predictions, confirming that the N-terminal region is very sensitive to changes in charge. For example, desMet D14N mutation destabilizes protein and decreases its calcium affinity. On the other hand, desMet E1M and desMet D37N substitutions increase the thermal stability and calcium affinity. The Met E1Q is characterized by a marked increase in protein stability, whereas desMet E7Q and desMet E11L display a slight increase in calcium affinity and thermal stability. Examination of the unfavorable energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue suggests that nature may have made an error with bovine alpha-lactalbumin from the viewpoint of stabilizing structure and conformation.
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