Journal: Journal of biomolecular NMR

Article Title: Evaluation of the tert -butyl group as a probe for NMR studies of macromolecular complexes

doi: 10.1007/s10858-021-00380-y

Figure Lengend Snippet: Chemical reactions and positions selected for tBu tagging in presynaptic proteins. a Diagram of the tBu group, with arrows illustrating the fast rotations that are expected to occur in the ps time scale around the bonds that link the methyl groups to the quaternary carbon and around the bond linking the quaternary carbon to the protein (P) if the tBu group does not pack against other atoms of the protein. b Diagrams of the chemical reactions used to attach tBu groups to protein cysteine residues. c Close up views of ribbon diagrams of the three structures of complexes between the Syt1 C2B domain (blue) and the SNARE four-helix bundle (green, red and yellow) that have been elucidated (Brewer et al., 2015; Zhou et al., 2015; Zhou et al., 2017) (PDB accession codes 2N1T, 5KJ7 and 5W5D, respectively). The structures were superimposed using the Syt1 C2B domain and only the C2B domain molecule corresponding to 5KJ7 is shown. The residues corresponding to the positions that were selected for mutation to cysteine for tagging with a tBu group (N319 and E346) are shown as orange spheres. Other side chains are shown by sticks. The Ca2+-binding sites of the C2B domain are located on the opposite end of the C2B domain β-sandwich, behind the plane of view, and are not observable in this diagram. d Ribbon diagram of the crystal structure of Cpx1(26-83) (pink) bound to the SNARE complex (synaptobrevin red, syntaxin-1 yellow, SNAP-25 green) (Chen et al., 2002) (PDB accession code 1KIL). The residues of syntaxin-1 and Cpx1(26-83) selected for mutation to cysteine and tagging with a tBu group (D214 and V61, respectively) are shown as purple and cyan spheres, respectively. Other side chains are shown by sticks.

Article Snippet: Constructs to express the following proteins or protein fragments were described previously: rat synaptobrevin-2 SNARE motif (residues 29–93), rat synaptobrevin-2 (residues 49–93), full-length rat synaptobrevin-2, human SNAP-25A fragments encoding its SNARE motifs (residues 11–82 and 141–203), full-length rat syntaxin-1A, rat syntaxin-1A (residues 183-288), rat syntaxin-1A (residues 191–253), rat syntaxin-1A (residues 2–253), rat synaptotagmin-1 C 2 B domain (residues 271–421), rat synaptotagmin-1 C 2 AB fragment (residues 140–421), rat complexin-1 (residues 26–83) and MSP1E3D1 (pMSP1E3D1 was a kind gift from Stephen Sligar; Addgene plasmid # 20066; http://n2t.net/addgene:20066 ; RRID:Addgene_20066) ( Brewer et al., 2015 ; Chen et al., 2006 ; Chen et al., 2008 ; Chen et al., 2002 ; Denisov et al., 2007 ; Ma et al., 2013 ; Rizo et al., 2006 ; Xu et al., 2013 ; Zhou et al., 2013 ).

Techniques: Mutagenesis, Binding Assay