title: An approach to quality management in structural biology: biophysical selection of proteins for successful crystallization.
abstract: Aggregation, incorrect folding and low stability are common obstacles for protein structure determination, and are often discovered at a very late state of protein production. In many cases, however, the reasons for failure to obtain diffracting crystals remain entirely unknown. We report on the contribution of systematic biophysical characterization to the success in structural determination of human proteins of unknown fold. Routine analysis using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) was employed to evaluate fold and stability of 263 purified protein samples (98 different human proteins). We found that FTIR-monitored temperature scanning may be used to detect incorrect folding and discovered a positive correlation between unfolding enthalpy measured with DSC and the size of small, globular proteins that may be used to estimate the quality of protein preparations. Furthermore, our work establishes that the risk of aggregation during concentration of proteins may be reduced through DLS monitoring. In summary, our study demonstrates that biophysical characterization provides an ideal tool to facilitate quality management for structural biology and many other areas of biological research.
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, D-10117 Berlin, Germany. frank.niesen@sgc.ox.ac.uk
authors.forename: Frank H
authors.initials: FH
authors.lastname: Niesen
authors.forename: Anja
authors.initials: A
authors.lastname: Koch
authors.forename: Ulf
authors.initials: U
authors.lastname: Lenski
authors.forename: Ulrich
authors.initials: U
authors.lastname: Harttig
authors.forename: Yvette
authors.initials: Y
authors.lastname: Roske
authors.forename: Udo
authors.initials: U
authors.lastname: Heinemann
authors.forename: Klaus Peter
authors.initials: KP
authors.lastname: Hofmann
mesh_terms: D001703:Biophysics / Q000379:methods*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005583:Fourier Analysis; D008027:Light; D010843:Pichia / Q000378:metabolism; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D011786:Quality Control; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012542:Scattering, Radiation; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D011506:Proteins