title: Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus.
abstract: Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment "invisible" and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA. Electronic address: kendra.frederick@utsouthwestern.edu.
authors.forename: Kendra K
authors.initials: KK
authors.lastname: Frederick
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Vladimir K
authors.initials: VK
authors.lastname: Michaelis
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Björn
authors.initials: B
authors.lastname: Corzilius
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Ta-Chung
authors.initials: TC
authors.lastname: Ong
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Angela C
authors.initials: AC
authors.lastname: Jacavone
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Griffin
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: lindquist_admin@wi.mit.edu.
authors.forename: Susan
authors.initials: S
authors.lastname: Lindquist
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000378:metabolism; D011328:Prions / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins