Exploiting elements of transcriptional machinery to enhance protein stability


Abstract

The correlation between protein structure and function is well established, yet the role stability/flexibility plays in protein function is being explored. Here, we describe an in vivo screen in which the thermal stability of a test protein is correlated directly to the transcriptional regulation of a reporter gene. The screen readout is independent of the function of the test protein, proteolytic resistance, solubility or propensity to aggregate indiscrimi- nately, and is thus dependent solely on the overall stability of the test protein. The system entails the use of an engineered chimeric construct that consists of three covalently linked domains; a constant N-terminal DNA- binding domain, a variable central test protein, and a constant C-terminal transcriptional activation domain. The test proteins are mutant variants of the β1 domain of streptococcal protein G that span fairly evenly a thermal stability range from as low as 38 °C to above 100 °C. When the chimeric construct contains a test variant of low thermal stability, the reporter gene is up-regulated to a greater extent relative to that of more stable/less flexible variants. A panel of nine Gβ1 mutant variants was used to benchmark the screen, and spectroscopic methods were employed to characterize the thermal and structural properties of each variant accurately. The screen was combined with in silico methods to interrogate a library of randomized variants for selection of mutants of greater structural integrity.

Submission Details

ID: N9MqCrW8

Submitter: Marie Ary

Submission Date: Feb. 15, 2017, 2:56 p.m.

Version: 1

Publication Details
Barakat NH;Barakat NH;Carmody LJ;Love JJ,J Mol Biol (2007) Exploiting elements of transcriptional machinery to enhance protein stability. PMID:17157872
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)