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1.  Cell-Free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility 
Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Non-conventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here we present a comparative study of the hepta-helical membrane protein bacteriorhodopsin in detergent micelles, amphipols and nanodiscs. Our results confirm that non-conventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are in principle accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguishes regions of bacteri-orhodopsin that mediate membrane/solvent contacts in the tested environments whereas the protein’s functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.
PMCID: PMC3595385  PMID: 23415558
2.  Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins 
Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent containing environments. While nanodiscs can offer a near native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin we show that our smaller nanodiscs can also be beneficial for the structural characterization of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for small to medium sized membrane proteins while still providing a functional environment. In addition to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of interactions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biological relevant interactions.
PMCID: PMC3566289  PMID: 23294159
Dynamics; Nanodiscs; NMR; OmpX; Structure
3.  Non-micellar systems for solution NMR spectroscopy of membrane proteins 
Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, non-micellar solubilization techniques, with a particular focus on their application to solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes.
PMCID: PMC2928847  PMID: 20570504
4.  Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy 
Journal of Biomolecular Nmr  2011;49(2):121-129.
It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all detectable species. The analysis was carried out using experimental data obtained during aggregation of the 10.4 kDa Crh protein, which has been shown to involve a partially unfolded intermediate state prior to aggregation. Based on a single real-time 2D 13C–13C transition spectrum, kinetic information about the refolding and aggregation step could be extracted. In addition, structural rearrangements associated with refolding are estimated and several different aggregation scenarios were compared to the experimental data.
PMCID: PMC3042102  PMID: 21253842
Aggregation; Kinetic; Solid-state NMR; Real-time spectroscopy; Crh
5.  Simultaneous use of solution, solid-state NMR and X-ray crystallography to study the conformational landscape of the Crh protein during oligomerization and crystallization 
We explore, using the Crh protein dimer as a model, how information from solution NMR, solid-state NMR and X-ray crystallography can be combined using structural bioinformatics methods, in order to get insights into the transition from solution to crystal. Using solid-state NMR chemical shifts, we filtered intra-monomer NMR distance restraints in order to keep only the restraints valid in the solid state. These filtered restraints were added to solid-state NMR restraints recorded on the dimer state to sample the conformational landscape explored during the oligomerization process. The use of non-crystallographic symmetries then permitted the extraction of converged conformers subsets. Ensembles of NMR and crystallographic conformers calculated independently display similar variability in monomer orientation, which supports a funnel shape for the conformational space explored during the solution-crystal transition. Insights into alternative conformations possibly sampled during oligomerization were obtained by analyzing the relative orientation of the two monomers, according to the restraint precision. Molecular dynamics simulations of Crh confirmed the tendencies observed in NMR conformers, as a paradoxical increase of the distance between the two β1a strands, when the structure gets closer to the crystallographic structure, and the role of water bridges in this context.
PMCID: PMC3170007  PMID: 21918624
structural bioinformatics; NMR structure calculation; ARIA; non-crystallographic symmetry; crystallographic ensemble refinement; molecular dynamics simulation
6.  Structural constraints for the Crh protein from solid-state NMR experiments 
Journal of Biomolecular Nmr  2008;40(4):239-250.
We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-008-9229-3) contains supplementary material, which is available to authorized users.
PMCID: PMC2579321  PMID: 18320329
Catabolite repression histidine-containing phosphocarrier protein (Crh); Distance constraints; MAS; 3D Protein structure; Solid-state NMR spectroscopy

Results 1-6 (6)