Figure 1.
Coverage map and CLUSTAL 2.1 multiple sequence alignment (after manual adjustments described in [24]) of L. stagnalis.
A. californica, and the extracellular domain (ECD) of human glycine receptor alpha1 subunit. Loops 7 and 9 of GlyR ECD have been mutated to obtain GlyBP (grey highlights on the alignment). Sequence highlighted in red cumulatively marks peptides whose mass ions are detected in control studies. As described in the text, tryptic fingerprinting of GlyBP gel slices typically resulted in 55–80% coverage.
Figure 2.
A) Structure of a single GlyBP subunit is shown in a ribbon representation. Colors represent mobility of individual residues in MD simulations. Mobility is measured by root mean squared fluctuations (RMSF) with respect to an average structure obtained in a steady-state dynamics. The coloring scheme is as follows: RMSF <0.8 Å - blue, RMSF range 0.8–1.3 Å - green, 1.3–1.5 Å –yellow, 1.5–1.8 Å – orange, RMSF> 1.8 Å - red. B) Conformational diversity of subunits within the GlyBP pentamer is shown by a structural superposition of average monomer structures (last 2 ns of the trajectory) color coded by root mean squared deviation between subunits. Front (outer side) and back (inner side) are shown in the left and right panels, respectively.
Figure 3.
Overview of experimental strategy used in MS/GlyBP modeling studies.
Figure 4.
Intramolecular crosslinks observed in GlyBP by MALDI-TOF MS analysis after crosslinking with DMS.
A) Representative mass spectrum of tryptic digest of excised monomeric GlyBP band. Mass peaks assigned as crosslinked peptides are labeled and further identified in Table 1. B) Average Cα-Cα Lys-Lys distances measured along the MD trajectory of assigned crosslinks are provided in the panel below. The calculation of distances is averaged over all 5 subunits/interfaces over the 2 ns long MD trajectory. The positions of the Lys residues in the modeled GlyBP (for simplicity, only the monomer is represented) are shown. The protein structure is shown in grey color in cartoon representation and Cα atoms of Lys residues are shown as colored spheres. C) The positions of Lys residues in the model of GlyBP for which we did not observe crosslinking (residue numbers in boxes): the panel shows the outer surface and the inner surfaces of a subunit - the latter also represented by space-filled model.
Table 1.
Intramolecular crosslinks identified by mass spectrometric studies of monomeric GlyBP bands.
Figure 5.
Intra-/inter-molecular crosslinks observed in GlyBP by MALDI-TOF MS analysis after crosslinking with DMS.
Representative mass spectrum of tryptic digest of excised higher molecular weight GlyBP band is shown in the top panel. Mass peaks assigned as crosslinked peptides are labeled and further identified in Table 2. Average Cα-Cα Lys-Lys distances measured along the MD trajectory of assigned crosslinks are provided in the panel below. The calculation of distances is averaged over all 5 subunits/interfaces over the 2 ns long MD trajectory. a and b indices distinguish adjacent GlyBP monomers in a pentamer. The positions of the Lys residues in two neighboring subunits of the GlyBP model are shown in bottom right. The protein structure is shown in grey and gold color in cartoon representation and Cα atoms of Lys residues are shown as colored spheres. * the range of distances reflect variations the average distance between subunits in the MD trajectory; while upper range distances are greater that the crosslinker length, the flexibility of the C loop in GlyBP brings the distances (underlined) well within the crosslinker arm length.
Table 2.
Intra-/Intermolecular crosslinks identified by mass spectrometric studies of higher order oligomeric GlyBP bands.