Zoya Ignatova* and Lila M. Gierascht
1. Introduction 356
2. Analysis of Effects of Osmolytes In Vivo 358
2.1. Culture growth and induction of osmolyte uptake in vivo 360
2.2. Partitioning into insoluble and soluble cell fractions 361
2.3. Variations in the effect of different osmolytes in vivo 362
3. Comparison of the Influence of Osmolytes In Vivo with That In Vitro 367
3.1. Expression and purification 367
3.2. In vitro stability measurements 368
3.3. In vitro aggregation kinetics 368
3.4. Different osmolytes have different effects on folding and aggregation in vitro 368
4. Conclusions 370 Acknowledgments 370 References 370
Nature has developed many strategies to ensure that the complex and challenging protein folding reaction occurs in vivo with adequate efficiency and fidelity for the success of the organism. Among the strategies widely employed in a huge range of species and cell types is the elaboration of small organic molecules called osmolytes that offset the potentially damaging effects of osmotic stress. While considerable knowledge has been gained in vitro regarding the influence of osmolytes on protein structure and folding, it is of great interest to probe the effects of osmolytes in cells. We have developed an in-cell fluorescent-labeling method that enables the study of protein stability and also protein aggregation in vivo. We utilize a genetically encoded tag called a tetra-Cys motif that binds specifically to a bis-arsenical fluorescein-based dye "FlAsH"; we inserted the tetra-Cys motif into a protein of interest in such a
* Max Planck Institute for Biochemistry, Martinsried, Germany
^ Departments of Biochemistry & Molecular Biology and Chemistry, University of Massachusetts, Amherst, Massachusetts
Methods in Enzymology, Volume 428 © 2007 Elsevier Inc.
ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)28021-8 All rights reserved.
way that the FlAsH signal reported on the state of folding or aggregation of the protein. Then, we designed protocols to assess how various osmolytes influence the stability and propensity to aggregate of our protein of interest. These are described here. Not only are there potential biotechnological applications of osmolytes in the quest to produce greater quantities of well-folded proteins, but also osmolytes may serve as tools and points of departure for therapeutic intervention in protein folding and aggregation diseases. Having in vivo methods to analyze how osmolytes affect folding and aggregation enhances our ability to further these goals greatly.
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