title: High-pressure SAXS study of folded and unfolded ensembles of proteins.
abstract: A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.
authors.affiliation: Fakultät Physik/DELTA, Technische Universität Dortmund, Dortmund, Germany.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Schroer
authors.forename: Michael
authors.initials: M
authors.lastname: Paulus
authors.forename: Christoph
authors.initials: C
authors.lastname: Jeworrek
authors.forename: Christina
authors.initials: C
authors.lastname: Krywka
authors.forename: Saskia
authors.initials: S
authors.lastname: Schmacke
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhai
authors.forename: D C Florian
authors.initials: DC
authors.lastname: Wieland
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Sahle
authors.forename: Michael
authors.initials: M
authors.lastname: Chimenti
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Royer
authors.forename: Bertrand
authors.initials: B
authors.lastname: Garcia-Moreno
authors.forename: Metin
authors.initials: M
authors.lastname: Tolan
authors.forename: Roland
authors.initials: R
authors.lastname: Winter
mesh_terms: D001274:Atmospheric Pressure; D002152:Calorimetry, Differential Scanning; D006863:Hydrogen-Ion Concentration; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D053838:Scattering, Small Angle; D012997:Solvents; D013696:Temperature; D014961:X-Ray Diffraction / Q000379:methods*
chemical_list: D050505:Mutant Proteins; D012997:Solvents; D008836:Micrococcal Nuclease