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MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.

G protein‐coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein‐stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope‐labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist‐induced activity of the receptor. Furthermore, this assay can be used as a readout when re‐introducing agonist‐dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively 19F‐labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis., Active or inactive? We used NMR to probe the structural states of stabilized neurotensin receptor (NTR1) variants in complex with different ligands by chemical modification of surface‐exposed cysteine residues. This protocol was further used to design NTR1 variants with wild‐type‐like conformational switching properties that can be produced in bacterial hosts.

Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization., Outer mitochondrial membrane permeabilization by pore‐forming Bcl2 proteins, including Bak, is crucial for apoptosis. Here, a high‐resolution structural characterization reveals large conformational changes of Bak during activation at the membrane surface.

G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals1,2. Although several structures have been solved for GPCR-G protein complexes3–17, structural studies of the complex in a physiological lipid membrane environment are lacking. Additionally, most previous studies required additional antibodies/nanobodies and/or engineered G proteins for complex stabilization. In the absence of a native complex structure, the underlying mechanism of G protein activation leading to GDP/GTP exchange remains unclear. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gαi1β1γ1 protein in two conformational states, resolved to 4.1 and 4.2 Å resolution. The structures were determined without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling under near native conditions.

Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein, allowing the preparation of a range of stepwise-smaller nanodiscs (6- to 8-nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope-labeling schemes required for working with large membrane protein systems in nanodiscs, and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (NOESY) spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12-24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.

Sequence variants of the microglial expressed TREM2 (triggering receptor expressed on myeloid cells 2) are a major risk factor for late onset Alzheimer's disease. TREM2 requires a stable interaction with DAP12 in the membrane to initiate signaling, which is terminated by TREM2 ectodomain shedding and subsequent intramembrane cleavage by γ‐secretase. To understand the structural basis for the specificity of the intramembrane cleavage event, we determined the solution structure of the TREM2 transmembrane helix (TMH). Caused by the presence of a charged amino acid in the membrane region, the TREM2‐TMH adopts a kinked structure with increased flexibility. Charge removal leads to TMH stabilization and reduced dynamics, similar to its structure in complex with DAP12. Strikingly, these dynamical features match with the site of the initial γ‐secretase cleavage event. These data suggest an unprecedented cleavage mechanism by γ‐secretase where flexible TMH regions act as key determinants of substrate cleavage specificity., NMR analyses of the TREM2 transmembrane helix in its native lipid environments reveal the structural basis for site‐specific intramembrane proteolysis of this disease‐associated microglial signaling receptor.

Sequence variants of the microglial expressed TREM2 (triggering receptor expressed on myeloid cells 2) are a major risk factor for late onset Alzheimer’s disease. TREM2 requires a stable interaction with DAP12 in the membrane to initiate signaling, which is terminated by TREM2 ectodomain shedding and subsequent intramembrane cleavage by γ-secretase. To understand the structural basis for the specificity of the intramembrane cleavage event, we determined the solution structure of the TREM2 transmembrane helix (TMH). Due to the presence of a charged amino acid in the membrane region the TREM2-TMH adopts a kinked structure with increased flexibility. Charge removal leads to TMH stabilization and reduced dynamics, similar to its structure in complex with DAP12. Strikingly, these dynamical features perfectly correlate with the site of the initial γ-secretase cleavage event. These data suggest an unprecedented cleavage mechanism by γ-secretase where flexible TMH regions are identified to initiate substrate cleavage.

A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs.

Membrane proteins are important players in signal transduction and the exchange of metabolites within or between cells. Thus, this protein class is the target of around 60 % of currently marketed drugs, emphasizing their essential biological role. Besides functional assays, structural and dynamical investigations on this protein class are crucial to fully understanding their functionality. Even though X-ray crystallography and electron microscopy are the main methods to determine structures of membrane proteins and their complexes, NMR spectroscopy can contribute essential information on systems that (a) do not crystallize and (b) are too small for EM. Furthermore, NMR is a versatile tool for monitoring functional dynamics of biomolecules at various time scales. A crucial aspect of such studies is the use of a membrane mimetic that resembles a native environment and thus enables the extraction of functional insights. In recent decades, the membrane protein NMR community has moved from rather harsh detergents to membrane systems having more native-like properties. In particular, most recently phospholipid nanodiscs have been developed and optimized mainly for solution-state NMR but are now also being used for solid-state NMR spectroscopy. Nanodiscs consist of a patch of a planar lipid bilayer that is encircled by different (bio-) polymers to form particles of defined and tunable size. In this review, we provide an overview of available membrane mimetics, including nanodiscs, amphipols and bicelles, that are suitable for high-resolution NMR spectroscopy and describe how these advanced membrane mimetics can facilitate NMR studies on the structure and dynamics of membrane proteins. Since the stability of membrane proteins depends critically on the chosen membrane mimetic, we emphasize the importance of a suitable system that is not necessarily developed for solution-state NMR applications and hence requires optimization for each membrane protein. However, lipid-based membrane mimetics offer the possibility of performing NMR experiments at elevated temperatures and studying ligand and partner protein complexes as well as their functional dynamics in a realistic membrane environment. In order to be able to make an informed decision during the selection of a suitable membrane system, we provide a detailed overview of the available options for various membrane protein classes and thereby facilitate this often-difficult selection process for a broad range of desired NMR applications. (C) 2019 Elsevier B.V. All rights reserved.

The voltage-dependent anion channel (VDAC) resides in the outer mitochondrial membrane and can adopt a closed or open configuration, most likely depending on whether the N-terminal segment (NTS) occupies the pore or protrudes into the cytoplasm. In this study, we calculate the free energy of releasing the NTS from the pore using molecular dynamics simulation. This is complicated by the flexible nature of the NTS, in particular its disordered structure in aqueous solution compared to the pore lumen. We carried out potential of mean force calculations using enhanced sampling or conformational restraints to address the conformational sampling problem. For the binding to the VDAC pore, two systems were considered, featuring either the native VDAC system or a modified system where the NTS is detached from the pore, that is, noncovalently bound in the pore lumen. The calculated free energies required to translocate the NTS from the pore into the solvent moiety are 83.8 or 74.3 kJ mol−1, respectively. The dissociation pathway in VDAC presents two in-pore minima, separated by a low free energy barrier and a membrane-bound intermediate state. Since we observe small changes in pore shape along the NTS dissociation pathway, we suggest that rigidification of the VDAC pore might impair NTS dissociation. The stability of the membrane-bound state of the VDAC NTS is confirmed by independent molecular dynamics simulations showing spontaneous membrane binding of a NTS-derived peptide as well as nuclear magnetic resonance experiments where chemical shift perturbations of the NTS-derived peptide evidence binding to phospholipid nanodiscs.

Phospholipid nanodiscs are a native-like membrane mimetic that is suitable for structural studies of membrane proteins. Although nanodiscs of different sizes exist for various structural applications, their thermal and long-term stability can vary considerably. Covalently circularized nanodiscs are a perfect tool to overcome these limitations. Existing methods for the production of circularized nanodiscs can be time-consuming and technically demanding. Therefore, an easy in vivo approach, in which circularized membrane scaffold proteins (MSPs) can be directly obtained from Escherichia coli culture, is reported herein. Nostoc punctiforme DnaE split-intein fusions with MSPs of various lengths are used and consistently provide circularized nanodiscs in high yields. With this approach, a large variety of circularized nanodiscs, ranging from 7 to 26 nm in diameter, that are suitable for NMR spectroscopy and electron microscopy (EM) applications can be prepared. These nanodiscs are superior to those of the corresponding linear versions in terms of stability and size homogeneity, which affects the quality of NMR spectroscopy data and EM experiments. Due to their long-term stability and homogeneity, the presented small circular nanodiscs are suited for high-resolution NMR spectroscopy studies, as demonstrated with two membrane proteins of 17 or 32 kDa in size. The presented method will provide easy access to circularized nanodiscs for structural studies of membrane proteins and for applications in which a defined and stable nanodisc size is required.

Purpose of Review Advances in technology have expanded telemedicine opportunities covering medical practice, research, and education. This is of particular importance in movement disorders (MDs), where the combination of disease progression, mobility limitations, and the sparse distribution of MD specialists increase the difficulty to access. In this review, we discuss the prospects, challenges, and strategies for telemedicine in MDs.Recent Findings Telemedicine for MDs has been mainly evaluated in Parkinson's disease (PD) and compared to in-office care is cost-effective with similar clinical care, despite the barriers to engagement. However, particular groups including pediatric patients, rare MDs, and the use of telemedicine in underserved areas need further research.Summary Interdisciplinary telemedicine and tele-education for MDs are feasible, provide similar care, and reduce travel costs and travel time compared to in-person visits. These benefits have been mainly demonstrated for PD but serve as a model for further validation in other movement disorders.

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique β-strand structure distinct from the conventional amyloid β-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner.

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A–TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1–DP1 module may serve as an Hsp70–client delivery system for the TPR2A–TPR2B–DP2 segment, which is required for client activation in vivo.

The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.

The molecular chaperone Hsp90 is required for the folding and activation of a large number of substrate proteins. These are involved in essential cellular processes ranging from signal transduction to viral replication. For the activation of its substrates, Hsp90 binds and hydrolyzes ATP, which is the key driving force for conformational conversions within the dimeric chaperone. Dimerization of Hsp90 is mediated by a C-terminal dimerization site. In addition, there is a transient ATP-induced dimerization of the two N-terminal ATP-binding domains. The resulting ring-like structure is thought to be the ATPase-active conformation. Hsp90 is a slow ATPase with a turnover number of 1 ATP/min for the yeast protein. A key question for understanding the molecular mechanism of Hsp90 is how ATP hydrolysis is regulated and linked to conformational changes. In this study, we analyzed the activation process structurally and biochemically with a view to identify the conformational limitations of the ATPase reaction cycle. We showed that the first 24 amino acids stabilize the N-terminal domain in a rigid state. Their removal confers flexibility specifically to the region between amino acids 98 and 120. Most surprisingly, the deletion of this structure results in the complete loss of ATPase activity and in increased N-terminal dimerization. Complementation assays using heterodimeric Hsp90 show that this rigid lid acts as an intrinsic kinetic inhibitor of the Hsp90 ATPase cycle preventing N-terminal dimerization in the ground state. On the other hand, this structure acts, in concert with the 24 N-terminal amino acids of the other N-terminal domain, to form an activated ATPase and thus regulates the turnover number of Hsp90.

SummarySelecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional 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 heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional 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 distinguish regions of bacteriorhodopsin 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.

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 biologically relevant interactions.

A huge variety of proteins are able to form fibrillar structures(1), especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand(2,3). Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal(4,5) and carboxy-terminal(6) domains. The N-terminal domain comprises a secretion signal, but further functions remain unassigned. The C-terminal domain was implicated in the control of solubility and fibre formation(7) initiated by changes in ionic composition(8,9) and mechanical stimuli known to align the repetitive sequence elements and promote beta-sheet formation(10-14). However, despite recent structural data(15), little is known about this remarkable behaviour in molecular detail. Here we present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre.

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-mu, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.

We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We sample the macrocyclic scaffold of the unbound ligand by parallel tempering simulations and dock the most populated conformations to yeast HSP90. Docking poses are then evaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of backbone (15)N provides an additional evaluation criteria. As a final test we check the binding modes against available structure-activity-relationships. We find that the most likely binding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol-HSP90 complex.

Disulfide bridges are one of the most important factors stabilizing the native structure of a protein. Whereas the basis for their stabilizing effect is well understood, their role in a protein folding reaction still seems to require further attention. We used the constant domain of the antibody light chain (CL), a representative of the ubiquitous immunoglobulin (Ig)-superfamily, to delineate the kinetic role of its single buried disulfide bridge. Independent of its redox state, the monomeric CL domain adopts a typical Ig-fold under native conditions and does not retain significant structural elements when unfolded. Interestingly, its folding pathway is strongly influenced by the disulfide bridge. The more stable oxidized protein folds via a highly structured on-pathway intermediate, whereas the destabilized reduced protein populates a misfolded off-pathway species on its way to the native state. In both cases, the formation of the intermediate species is shown to be independent of the isomerization state of the Tyr141-Pro142 bond. Our results demonstrate that the internal disulfide bridge in an antibody domain restricts the folding pathway by bringing residues of the folding nucleus into proximity thus facilitating the way to the native state.

The interaction between the cell-penetrating peptide (CPP) penetratin and different membrane mimetic environments has been investigated by two different NMR methods: 15N spin relaxation and translational diffusion. Diffusion coefficients were measured for penetratin in neutral and in negatively charged bicelles of different size, in sodium dodecyl sulfate micelles (SDS), and in aqueous solution. The diffusion coefficients were used to estimate the amount of free and bicelle/micelle-bound penetratin and the results revealed that penetratin binds almost fully to all studied membrane mimetics. 15N relaxation data for three sites in penetratin were interpreted with the model-free approach to obtain overall and local dynamics. Overall correlation times for penetratin were in agreement with findings for other peptides of similar size in the same solvents. Large differences in order parameters were observed for penetratin in the different membrane mimetics. Negatively charged surfaces were seen to restrict motional flexibility, while a more neutral membrane mimetic did not. This indicates that although the peptide binds to both bicelles and SDS micelles, the interaction between penetratin and the various membrane mimetics is different.