Site-specific amino acid alterations in Ca2+ binding domains in calmodulin impair activation of RBC Ca(2+)-ATPase

Evolution drives changes in a protein's sequence over time. The extent to which these changes in sequence lead to shifts in the underlying preference for each amino acid at each site is an important question with implications for comparative sequence-analysis methods such as molecular phylogenetics. To quantify the extent that site-specific amino-acid preferences shift during evolution, we performed deep mutational scanning on two homologs of human influenza nucleoprotein with 94% amino-acid identity. We found that only a modest fraction of sites exhibited shifts in amino-acid preferences that exceeded the noise in our experiments. Furthermore, even among sites that did exhibit detectable shifts, the magnitude tended to be small relative to differences between non-homologous proteins. Given the limited change in amino-acid preferences between these close homologs, we tested whether our measurements could inform site-specific substitution models that describe the evolution of nucleoproteins from more diverse influenza viruses. We found that site-specific evolutionary models informed by our experiments greatly outperformed non-site-specific alternatives in fitting phylogenies of nucleoproteins from human, swine, equine, and avian influenza. Combining the experimental data from both homologs improved phylogenetic fit, partly because measurements in multiple genetic contexts better captured the evolutionary average of the amino-acid preferences for sites with shifting preferences. Our results show that site-specific amino-acid preferences are sufficiently conserved that measuring mutational effects in one protein provides information that can improve quantitative evolutionary modeling of nearby homologs.

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Bioconjugates of Intelligent Polymers and Recognition Proteins for Use in Diagnostics and Affinity Separations

Clinical Chemistry ◽

10.1093/clinchem/46.9.1478 ◽

2000 ◽

Vol 46 (9) ◽

pp. 1478-1486 ◽

Cited By ~ 139

Author(s):

Allan S Hoffman

Keyword(s):

Amino Acid ◽

Protein A ◽

Acid Sites ◽

Genetically Engineered ◽

Smart Polymers ◽

Amino Groups ◽

Site Specific ◽

Specific Amino Acid ◽

Random Polymer ◽

Intelligent Polymers

Abstract Polymers that respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties are often called “intelligent” or “smart” polymers. We have conjugated these polymers to different recognition proteins, including antibodies, protein A, streptavidin, and enzymes. These bioconjugates have been prepared by random polymer conjugation to lysine amino groups on the protein surface, and also by site-specific conjugation of the polymer to specific amino acid sites, such as cysteine sulfhydryl groups, that are genetically engineered into the known amino acid sequence of the protein. We have conjugated several different smart polymers to streptavidin, including temperature-, pH-, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed here.

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