Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering
Abstract
In this paper, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.
- Authors:
-
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
- Monash Univ., Victoria (Australia)
- UT Southwestern Medical Center, Dallas, TX (United States)
- Integral Molecular, Philadelphia, PA (United States)
- Integral Molecular, Philadelphia Pennsylvania
- Publication Date:
- Research Org.:
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1240402
- Report Number(s):
- LA-UR-14-28954
Journal ID: ISSN 0887-3585
- Grant/Contract Number:
- GM063210; CBS.MEDBIO.04.10.LA.008; 20120449ER; AC52-06NA25396
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Proteins
- Additional Journal Information:
- Journal Volume: 83; Journal Issue: 7; Journal ID: ISSN 0887-3585
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; protein engineering; GFP structure; X-ray crystallography; thermal green protein; thermal stability; eCGP123; structure-guided mutagenesis
Citation Formats
Close, Devin W., Paul, Craig Don, Langan, Patricia S., Wilce, Matthew C. J., Traore, Daouda A. K., Halfmann, Randal, Rocha, Reginaldo C., Waldo, Geoffery S., Payne, Riley J., Rucker, Joseph B., Prescott, Mark, and Bradbury, Andrew R. M. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering. United States: N. p., 2015.
Web. doi:10.1002/prot.24699.
Close, Devin W., Paul, Craig Don, Langan, Patricia S., Wilce, Matthew C. J., Traore, Daouda A. K., Halfmann, Randal, Rocha, Reginaldo C., Waldo, Geoffery S., Payne, Riley J., Rucker, Joseph B., Prescott, Mark, & Bradbury, Andrew R. M. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering. United States. https://doi.org/10.1002/prot.24699
Close, Devin W., Paul, Craig Don, Langan, Patricia S., Wilce, Matthew C. J., Traore, Daouda A. K., Halfmann, Randal, Rocha, Reginaldo C., Waldo, Geoffery S., Payne, Riley J., Rucker, Joseph B., Prescott, Mark, and Bradbury, Andrew R. M. Fri .
"Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering". United States. https://doi.org/10.1002/prot.24699. https://www.osti.gov/servlets/purl/1240402.
@article{osti_1240402,
title = {Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering},
author = {Close, Devin W. and Paul, Craig Don and Langan, Patricia S. and Wilce, Matthew C. J. and Traore, Daouda A. K. and Halfmann, Randal and Rocha, Reginaldo C. and Waldo, Geoffery S. and Payne, Riley J. and Rucker, Joseph B. and Prescott, Mark and Bradbury, Andrew R. M.},
abstractNote = {In this paper, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.},
doi = {10.1002/prot.24699},
journal = {Proteins},
number = 7,
volume = 83,
place = {United States},
year = {Fri May 08 00:00:00 EDT 2015},
month = {Fri May 08 00:00:00 EDT 2015}
}
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