Effects of macromolecular crowding and small ions on the folding, structure, and stability of Desulfovibrio desufuricans flavodoxin

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dc.contributor.advisorWittung-Stafshede, Pernillaen_US
dc.creatorStagg, Lorenen_US
dc.date.accessioned2011-07-25T01:51:04Zen_US
dc.date.available2011-07-25T01:51:04Zen_US
dc.date.issued2010en_US
dc.description.abstractThe intracellular environment in which most proteins fold and function contains a range of biomolecules that results in significant volume exclusion, thus contrasting to the dilute buffer conditions common to most in vitro studies. In addition to intracellular macromolecular crowding, cells are ionic in nature, and although the Hofmeister series of ions has its origin in a work from 1888, much is still unclear concerning how small, charged ions affect protein properties. This thesis summarizes in vitro work assessing the effects of macromolecular crowding and small ions on the biophysical properties of a model protein -- Desulfovibrio desulfuricans flavodoxin. Flavodoxin is a small (15.7 kDa), single domain, cytoplasmic protein with alpha-helical and parallel beta-sheet secondary structural elements arranged in one of the five most common protein folds (the flavodoxin-like fold). Using a range of biophysical/spectroscopic methods (e.g., circular dichroism (CD), fluorescence, calorimetry, stopped-flow mixing) along with synthetic crowding agents (e.g., Ficoll and dextran), I have found that macromolecular crowding increases the secondary structural content of folded flavodoxin (toward that found in the crystal structure), increases flavodoxin thermal stability, and affects both the accumulation of a misfolded intermediate and the rate of proper protein folding. Collaborative in silico simulations employing Go-like modeling of apoflavodoxin in the presence of large, inert crowding agents agrees with my in vitro work and provides structural and mechanistic information with residue-specific resolution. We also found that small cations and anions in physiologically relevant concentrations (≤ 250 mM) increase flavodoxin thermal stability significantly. Both cations and anions in higher concentrations (300 mM-.75 M) affect oppositely charged proteins similarly suggesting that surface electrostatic charge plays only a minor role in mediating ionic effects on protein thermal stability. At all ion concentrations, ionic effects on protein stability are correlated to ion hydration (and thus the Hofmeister series). Our work suggests a dominant role for the peptide bond in coordinating ions at higher concentrations. This thesis work suggests that the crowded and ionic nature of the intracellular milieu can elicit changes to the structure, dynamics, stability, and folding mechanism of proteins which may not be captured in vitro using dilute buffer conditions.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoTHESIS BIOCHEM. 2010 STAGGen_US
dc.identifier.citationStagg, Loren. "Effects of macromolecular crowding and small ions on the folding, structure, and stability of Desulfovibrio desufuricans flavodoxin." (2010) Diss., Rice University. <a href="https://hdl.handle.net/1911/61970">https://hdl.handle.net/1911/61970</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/61970en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectBiophysicsen_US
dc.titleEffects of macromolecular crowding and small ions on the folding, structure, and stability of Desulfovibrio desufuricans flavodoxinen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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