title: Effects of protein stabilizing agents on thermal backbone motions: a disulfide trapping study.
abstract: Chemical stabilizers are widely used to enhance protein stability, both in nature and in the laboratory. Here, the molecular mechanism of chemical stabilizers is studied using a disulfide trapping assay to measure the effects of stabilizers on thermal backbone dynamics in the Escherichia coli galactose/ glucose binding protein. Two types of backbone fluctuations are examined: (a) relative movements of adjacent surface alpha-helices within the same domain and (b) interdomain twisting motions. Both types of fluctuations are significantly reduced by all six stabilizers tested (glycerol, sucrose, trehalose, L-glucose, D-glucose, and D-galactose), and in each case larger amplitude motions are inhibited more than smaller ones. Motional inhibition does not require a high-affinity stabilizer binding site, indicating that the effects of stabilizers are nonspecific. Overall, the results support the theory that effective stabilizing agents act by favoring the most compact structure of a protein, thereby reducing local backbone fluctuations away from the fully folded state. Such inhibition of protein backbone dynamics may be a general mechanism of protein stabilization in extreme thermal or chemical environments.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Butler
authors.forename: J J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D005690:Galactose / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011489:Protein Denaturation; D015202:Protein Engineering
chemical_list: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004220:Disulfides; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005690:Galactose