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While reverse genetics and functional genomics have long affirmed the role of individual mutations in determining protein function, there have been fewer studies addressing how larger-scaled changes in protein sequences, such as in entire modular segments, influence protein function and evolution. Given how recombination can reassort protein sequences, these types of changes may play an underappreciated role in how novel protein functions evolve in nature. Such studies could aid our understanding of whether certain organismal phenotypes related to protein function-such as growth in the presence or absence of an antibiotic-are robust with respect to the identity of certain modular segments. In this study, we combine molecular genetics with biochemical and biophysical methods to gain a better understanding of protein modularity in dihydrofolate reductase, an enzyme target of antibiotics also widely used as a model for protein evolution. We replace an integral α-helical segment of Escherichia coli DHFR with segments from a number of different organisms (many non-microbial) and examine how these chimeric enzymes affect organismal phenotypes (e.g. resistance to an antibiotic) as well as biophysical properties of the enzyme (e.g. thermostability). We find that organismal phenotypes and enzyme properties are highly sensitive to the identity of DHFR modules, and that this chimeric approach can create enzymes with diverse biophysical characteristics. This article is protected by copyright. All rights reserved.
This article was published in the following journal.
Name: Protein science : a publication of the Protein Society
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