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Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, α-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the α-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an α-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Membrane proteins in their native cellular membranes are accessible by dynamic nuclear polarization magic angle spinning solid-state NMR spectroscopy without the need of purification and reconstitution (see picture). Dynamic nuclear polarization is essential to achieve the required gain in sensitivity to observe the membrane protein of interest., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Membrane traffic between the trans-Golgi network (TGN) and endosomes is mediated in part by the assembly of clathrin-AP-1 adaptor complex-coated vesicles. This process involves multiple accessory proteins that directly bind to the ear domain of AP-1gamma via degenerate peptide motifs that conform to the consensus sequence diameterG(P/D/E)(diameter/L/M) (with diameter being a large hydrophobic amino acid). Recently, gamma-BAR (hereafter referred to as Gadkin for reasons explained below) has been identified as a novel AP-1 recruitment factor involved in AP-1-dependent endosomal trafficking of lysosomal enzymes. How precisely Gadkin interacts with membranes and with AP-1gamma has remained unclear. Here we show that Gadkin is an S-palmitoylated peripheral membrane protein that lacks stable tertiary structure. S-Palmitoylation is required for the recruitment of Gadkin to TGN/endosomal membranes but not for binding to AP-1. Furthermore, we identify a novel subtype of AP-1-binding motif within Gadkin that specifically associates with the gamma1-adaptin ear domain. Mutational inactivation of this novel type of motif, either alone or in combination with three more conventional AP-1gamma binding peptides, causes Gadkin to mislocalize to the plasma membrane and interferes with its ability to render AP-1 brefeldin A-resistant, indicating its physiological importance. Our studies thus unravel the molecular basis for Gadkin-mediated AP-1 recruitment to TGN/endosomal membranes and identify a novel subtype of the AP-1-binding motif.

Endosomes and endosomal vesicles (EVs) rapidly move along cytoskeletal filaments allowing them to exchange proteins and lipids between different endosomal compartments, lysosomes, the trans-Golgi network (TGN), and the plasma membrane. The precise mechanisms that connect membrane traffic between the TGN and perinuclear endosomal compartments with motor-protein driven transport have largely remained elusive. Here we show that Gadkin (also termed gamma-BAR), a peripheral membrane protein localized to the TGN and to TGN-derived EVs, directly associates with the clathrin adaptor AP-1 and with the motor protein kinesin KIF5, thereby potentially regulating EV dynamics. Gadkin overexpression induced the dispersion of transferrin (Tf)- and Rab4-positive EVs to the cell periphery, whereas KIF5B-depleted cells displayed a perinuclear concentration. Functional experiments suggest that the role of Gadkin as a regulator of endosomal membrane traffic critically depends on complex formation with both AP-1 and KIF5. Our data thus provide a direct molecular link between TGN-derived EVs and the microtubule-based cytoskeleton.

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken alpha-spectrin SH3 domain (62 residues), alphaB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Calpha, Cbeta, C' and N resonances in the core domain of alphaB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).

Formin homology 1 (FH1), is a long proline-rich region of formins, shown to bind to five WW containing proteins named formin binding proteins (FBPs). FH1 has several potential binding regions but only the PPLPx motif and its interaction with FBP11WW1 has been characterized structurally. To detect whether additional motifs exist in FH1, we synthesized five peptides and investigated their interaction with FBP28WW2, FBP11WW1 and FBP11WW2 domains. Peptides of sequence PTPPPLPP (positive control), PPPLIPPPP and PPLIPPPP (new motifs) interact with the domains with micromolar affinity. We observed that FBP28WW2 and FBP11WW2 behave differently from FBP11WW1 in terms of motif selection and affinity, since they prefer a doubly interrupted proline stretch of sequence PPLIPP. We determined the NMR structure of three complexes involving the FBP28WW2 domain and the three ligands. Depending on the peptide under study, the domain interacts with two proline residues accommodated in either the XP or the XP2 groove. This difference represents a one-turn displacement of the domain along the ligand sequence. To understand what drives this behavior, we performed further structural studies with the FBP11WW1 and a mutant of FBP28WW2 mimicking the XP2 groove of FBP11WW1. Our observations suggest that the nature of the XP2 groove and the balance of flexibility/rigidity around loop 1 of the domain contribute to the selection of the final ligand positioning in fully independent domains. Additionally, we analyzed the binding of a double WW domain region, FBP11WW1-2, to a long stretch of FH1 using fluorescence spectroscopy and NMR titrations. With this we show that the presence of two consecutive WW domains may also influence the selection of the binding mode, particularly if both domains can interact with consecutive motifs in the ligand. Our results represent the first observation of protein-ligand recognition where a pair of WW and two consecutive motifs in a ligand participate simultaneously.

Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.

Pex13p is an essential component of the peroxisomal protein import machinery and interacts via its C-terminal SH3 domain with the type II SH3-ligand Pex14p and the non-PXXP protein Pex5p. We report the solution structure of the SH3 domain of Pex13p from Saccharomyces cerevisiae and the identification of a novel-binding pocket, which binds a non-PXXP-peptide representing the binding site of Pex5p. Chemical shift assays revealed the binding sites for Pex5p and Pex14p ligand peptides to be distinct and spatially separated. Competition assays demonstrated that the two ligand peptides can bind simultaneously to the SH3 domain.

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.