Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over‐looked physiological factors

Autor: Chamitha J. Weeramange, Liskin Swint-Kruse, Kristen M. Schwingen, Joshua J. Riepe, Sudheer Tungtur
Rok vydání: 2019
Předmět:
Zdroj: Protein Sci
ISSN: 1469-896X
0961-8368
DOI: 10.1002/pro.3695
Popis: To bridge biological and biochemical disciplines, the relationship between in vitro protein biochemical function and in vivo activity must be established. Such studies can (a) help determine whether properties measured in simple, dilute solutions extrapolate to the complex in vivo conditions and (b) illuminate cryptic biological factors that are new avenues for study. We have explored the in vivo–in vitro relationship for chimeras built from LacI/GalR transcription regulators. In prior studies of individual chimeras, amino acid changes that altered in vitro DNA binding affinity exhibited correlated changes in in vivo transcription repression. However, discrepancies arose when the two datasets were compared to each other: Although their DNA binding domains were identical and their in vitro binding affinities spanned the same range, their in vivo repression ranges differed by >50‐fold. Here, we determined that the presence of endogenous ligand for one chimera further exacerbated the offset, but that different abilities to simultaneously bind and “loop” two DNA operators resolves the discrepancy. Indeed, results suggest that the lac operon can be looped by even weakly interacting repressor dimers. For looping‐competent repressors, we measured in vitro binding to the secondary operator. Surprisingly, this was largely insensitive to amino acid changes in the repressor protein, which reflects altered specificity; this supports the emerging view that the locations of specificity determining positions can be unique to each protein homolog. In aggregate, this work illustrates how a comparative approach can enrich understanding of the in vivo–in vitro relationship and suggest unexpected avenues for future study.
Databáze: OpenAIRE
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