Use of NMR to study serpin function.
Two-dimensional heteronuclear [1H,15N] single quantum correlation NMR spectra of serpins show dramatic changes between native and loop-inserted conformations, making them very sensitive reporters of the serpin conformation. Much of the spectral overlap that arises when all amides are 15N labelled can be removed by use of selective labelling of a single type of amino acid, such as alanine. The method allows ready determination of whether loop insertion is present, and to what extent, as well as providing information on motional freedom of components of the complex and of the reactive center loop. With label introduced separately into the proteinase, information can also be obtained on the conformational changes brought about in that moiety by complex formation. In addition, with the use of cryoprobes, high field spectrometers, TROSY-based signal detection and deuteration, samples as small as 1-2mg can easily be examined, making it applicable to a wide range of serpins, including those that can only be expressed in mammalian cells.
Gettins
Peter G W
PG
Department of Biochemistry and Molecular Genetics, M/C 669, University of Illinois at Chicago, 900 South Ashland, Chicago, IL 60607, USA. pgettins@uic.edu
Backovic
Marija
M
Peterson
Francis C
FC
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007553:Isotope Labeling / Q000379:methods; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis; D015843:Serpins / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000378:metabolism
D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins; D015843:Serpins; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; C038915:alpha 1-antitrypsin Pittsburgh; D014357:Trypsin