Proteins perform their functions in cells where macromolecular solutes can reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules will stabilize globular proteins because the native state of a globular protein occupies less space than the denatured state. The idea that volume exclusion should fold globular proteins is tested in Escherichia coli by using protein L Kx7E, a 7-kDa unstable globular protein variant of wild-type protein L. This variant requires high concentrations of monovalent salts to fold. The standard unfolding free energy of Kx7E in the absence of NaCl is −1.0 kcal/mol as determined by using in vitro 1H-15N nuclear magnetic resonance spectroscopy. In-cell nuclear magnetic resonance spectroscopy with 19F-labeled Kx7E is used to show that the crowded cytoplasm of E. coli cannot overcome even this modest free energy deficit and fold the variant. The data are consistent with the idea that non-specific interactions between cytoplasmic components can overcome the excluded volume effect. Evidence for these interactions is provided by the observation that adding simple salts folds Kx7E in dilute solution, but increasing the salt concentration inside E. coli does not fold the variant.
An attempt was made to acquire nuclear magnetic resonance spectra of the intrinsically disordered protein &agr;-synuclein and the green fluorescent protein inside insect cells. Infection of Spodoptera frugiperda insect cells with recombinant baculoviruses containing a late basic protein promoter produces sufficient recombinant protein while maintaining cell viability for spectral acquisition. However, incorporation of the nuclear magnetic resonance active isotopes 15N or 19F into the recombinant proteins through the addition of 15NH4Cl or 5-fluorotryptophan to the cell culture media proved unsuccessful. The similarity of the spectra for &agr;-synuclein, green fluorescent protein, and a fusion protein suggests isotope incorporation into intracellular metabolites. Thus, protein nuclear magnetic resonance spectra cannot be acquired in insect cells by using these experimental conditions.
|Advisor:||Pielak, Gary J.|
|Commitee:||Jarstfer, Michael B., Lee, Andrew, Redinbo, Matthew, Tripathy, Ashutosh|
|School:||The University of North Carolina at Chapel Hill|
|Department:||Biochemistry & Biophysics|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 72/11, Dissertation Abstracts International|
|Keywords:||Globular proteins, In-cell NMR, Protein unfolding, Volume effect|
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