Untitled

Abstract

Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

“…S3). This underlines the importance of a native lipid environment and supports arguments that the dimer only assumes an asymmetric conformation in its active state within the membrane (22,47,54,60).…”

“…6). Peak doubling has been observed previously by solid state NMR and structural significance could be assigned in cases with large chemical shift changes (Ͼ1.5 ppm), whereas smaller differences were suggested to arise from motions in the millisecond range (47,48). Hydrogen/deuterium exchange infrared spectroscopy is commonly used to judge relative membrane protein mobility.…”

Heteronuclear proton assisted recoupling

“…14(a)]. The spectrum exhibits a large number of additional 15 N- 13 C correlations compared to the intra-residue 2D NCACB reported in Bockmann et al 31 Using the x-ray structure of the Crh protein (PDB entry: 1MU4) (Ref. 30) and a distance cut-off of about 6 Å, we can classified the cross peaks in sequential, medium-range and long-range 15 N-13 C contacts.…”

“…Figure 14(a) shows the 2D 15 N-13 C correlation spectrum of the Crh protein, a dimeric crystalline model protein of 2*85 residues, with a known x-ray structure 30 and solid-state NMR chemical shifts. 31 The spectrum was recorded using 10 ms of PAIN-CP mixing time (ω 0H /2π = 750 MHz, ω r /2π = 20 kHz). The spectrum displays a large number of cross peaks, including many long distance contacts.…”

Polarisationstransfer über die Wasser‐Protein‐Grenzfläche im Festkörper

“…[8,11,18] Fast alle Kreuzsignale verlieren beim Absenken der Probentemperatur an Intensität, mit Ausnahme der His-Resonanzen (alle im 6 His-Tag), die wegen der langsameren Moleküldynamik bei tieferen Temperaturen, die den Polarisationstransfer durch Kreuzpolarisation (CP) begünstigen, zunächst an Intensität gewinnen. Die meisten positiven Signale sind eindeutig auf chemischen Austausch zurückzuführen (Abbildung 2 b, links).…”