Selective deuteration of tryptophan and methionine residues in maltose binding protein: a model system for neutron scattering.
We describe methods that have been developed within the ILL-EMBL Deuteration Laboratory for the production of maltose binding protein (MBP) that has been selectively labelled either with deuterated tryptophan or deuterated methionine (single labelling), or both (double labelling). MBP is used as an important model system for biophysical studies, and selective labelling can be helpful in the analysis of small-angle neutron scattering (SANS) data, neutron reflection (NR) data, and high-resolution neutron diffraction data. The selective labelling was carried out in E. coli high-cell density cultures using auxotrophic mutants in minimal medium containing the required deuterated precursors. Five types of sample were prepared and studied: (1) unmodified hydrogenated MBP (H-MBP), (2) perdeuterated MBP (D-MBP), (3) singly labelled MBP with the tryptophan residues deuterated (D-trp MBP), (4) singly labelled MBP with methionine residues deuterated (D-met MBP) and (5) doubly labelled MBP with both tryptophan and methionine residues deuterated (D-trp/met MBP). Labelled samples were characterised by size exclusion chromatography, gel electrophoresis, light scattering and mass spectroscopy. Preliminary small-angle neutron scattering (SANS) experiments have also been carried out and show measurable differences between the SANS data recorded for the various labelled analogues. More detailed SANS experiments using these labelled MBP analogues are planned; the degree to which such data could enhance structure determination by SANS is discussed.
Laux
Valerie
V
ILL-EMBL Deuteration Laboratory, Partnership for Structural Biology, Institut Laue Langevin, 38042, Grenoble Cedex 9, France.
Callow
Phil
P
Svergun
Dmitri I
DI
Timmins
Peter A
PA
Forsyth
V Trevor
VT
Haertlein
Michael
M
D002352:Carrier Proteins / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry*; D058619:Maltose-Binding Proteins; D008715:Methionine / Q000737:chemistry*; D033363:Neutron Diffraction / Q000379:methods*; D011485:Protein Binding; D053838:Scattering, Small Angle; D013194:Staining and Labeling / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D014364:Tryptophan; D008715:Methionine; D003903:Deuterium