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G protein-coupled receptors (GPCR) activate numerous intracellular signaling pathways. The oligomerization properties of GPCRs, and hence their cellular functions, may be modulated by various components within the cell membrane (such as the presence of cholesterol). Modulation may occur directly via specific interaction with the GPCR or indirectly by affecting the physical properties of the membrane. Here, we use pulsed Q-band double electron–electron resonance (DEER) spectroscopy to probe distances between R1 nitroxide spin labels attached to Cys163 and Cys344 of the β 1 -adrenergic receptor (β 1 AR) in n -dodecyl-β-D-maltoside micelles upon titration with two soluble cholesterol analogs, cholesteryl hemisuccinate (CHS) and sodium cholate. The former, like cholesterol, inserts itself into the lipid membrane, parallel to the phospholipid chains; the latter is aligned parallel to the surface of membranes. Global quantitative analysis of DEER echo curves upon titration of spin-labeled β 1 AR with CHS and sodium cholate reveal the following: CHS binds specifically to the β 1 AR monomer at a site close to the Cys163-R1 spin label with an equilibrium dissociation constant K D CHS ~1.4 ± 0.4 mM. While no direct binding of sodium cholate to the β 1 AR receptor was observed by DEER, sodium cholate induces specific β 1 AR dimerization ( K D cholate ~35 ± 6 mM and a Hill coefficient n ~ 2.5 ± 0.4) with intersubunit contacts between transmembrane helices 1 and 2 and helix 8. Analysis of the DEER data obtained upon the addition of CHS to the β 1 AR dimer in the presence of excess cholate results in dimer dissociation with species occupancies as predicted from the individual K D values.

The global motions of ubiquitin, a model protein, on the surface of anisotropically tumbling 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-racglycerol) (POPG):1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles are described. The shapes of POPG:DHPC bicelles prepared with high molar ratios q of POPG to DHPC can be approximated by prolate ellipsoids, with the ratio of ellipsoid dimensions and dimensions themselves increasing with higher values of q. Adaptation of the nuclear magnetic resonance (NMR) relaxation-based approach that we previously developed for interactions of ubiquitin with spherical POPG liposomes (Ceccon, A. et al. J. Am. Chem. Soc. 2016, 138, 5789–5792) allowed us to quantitatively analyze the variation in lifetime line broadening of NMR signals (ΔR(2)) measured for ubiquitin in the presence of q = 2 POPG:DHPC bicelles and the associated transverse spin relaxation rates (R(2,B)) of bicelle-bound ubiquitin. Ubiquitin, transiently bound to POPG:DHPC bicelles, undergoes internal rotation about an axis orthogonal to the surface of the bicelle and perpendicular to the principal axis of its rotational diffusion tensor on the low microsecond time scale (~3 μs), while the rotation axis itself wobbles in a cone on a submicrosecond time scale (≤ 500 ns).

The RHO gene encodes the G‐protein‐coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light‐activated state by solution and MAS‐NMR spectroscopy. We found two long‐lived dark states for the G90D mutant with the 11‐cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11‐cis retinal caused by the mutation‐induced distortion on the salt bridge formation in the binding pocket. Time‐resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle., Rhodopsin is the major dim light receptor in vertebrate eyes. Numerous mutations associated with impaired visual cycles are known. The G90D mutation leads to a constitutively active mutant form of rhodopsin that causes congenital stationary night blindness (CSNB). We investigated the consequences of this mutation on the visual cycle, both in terms of structural aspects and dynamic changes.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.

Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1 ) with high affinity (app. KD = 0.76 µm± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1 . Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1 .

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.

The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.

The conformational propensity of amino acid residues is determined by an intricate balance of peptide-solvent and solvent-solvent interactions. To explore how the systematic replacement of water by a cosolvent affects the solvation of both the amino acid backbone and side chains, we performed a combined vibrational spectroscopy and NMR study of cationic glycylalanylglycine (GAG) in different ethanol/water mixtures of between 0 and 42 mol percent ethanol. Classical model peptide N'-methylacetamide was used as a reference system to probe solvent-induced spectroscopic changes. The alanine residue of GAG in water is known to exhibit a very high propensity for polyproline II (pPII). Adding up to 30 mol % ethanol at room temperature leads only to minor changes in the Ramachandran distribution of alanine, which mostly changes within the individual conformational subspaces. A further increase in the ethanol fractions leads to a destabilization of pPII and a stabilization of β-strand conformations. At higher temperatures, different degrees of enthalpy-entropy compensations lead to a much stronger influence of ethanol on the peptide's conformational distribution. Ethanol-induced changes in chemical shifts and amide I wavenumbers strongly suggest that ethanol replaces water preferentially in the solvation shell of the polar C-terminal peptide group and of the alanine side chain, whereas the N-terminal group remains mostly hydrated. Furthermore, we found that ethanol interacts more strongly with the peptide if the latter adopts β-strand conformations. This leads to an unusual positive temperature coefficient for the chemical shift of the C-terminal amide proton. Our data suggests a picture in which GAG eventually accumulates at water-ethanol interfaces if the ethanol fractions exceed 0.3, which explains why the further addition of ethanol eventually causes self-aggregation and the subsequent formation of a hydrogel.

Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.

Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution‐state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition., Reading expands horizons: Structural analysis of small proteins encoded by previously overlooked small open reading frames (sORFs) is achieved by using NMR spectroscopy. A protocol is established for fast NMR spectroscopy structure screening, including optimized sample preparation for systems of this size (14–78 aa), and determination of the structural conformations adopted by the systems.

To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we combined vibrational and 2D NMR spectroscopy to obtain the conformational distributions of selected "GxyG" host-guest peptides in aqueous solution: GDyG, GSyG, GxLG, GxVG, where x/y=A, K, L, V. Large changes of conformational propensities were observed due to nearest-neighbor interactions, at variance with the isolated pair hypothesis. We found that protonated aspartic acid and serine lose their above-the-average preference for turn-like structures in favor of polyproline II (pPII) populations in the presence of neighbors with bulky side chains. Such residues also decrease the above-the-average pPII preference of alanine. These observations suggest that the underlying mechanism involves a disruption of the hydration shell. Thermodynamic analysis of (3) J(H(N) ,H(α) ) (T) data for each x,y residue reveals that modest changes in the conformational ensemble masks larger changes of enthalpy and entropy governing the pPII↔β equilibrium indicating a significant residue dependent temperature dependence of the peptides' conformational ensembles. These results suggest that nearest-neighbor interactions between unlike residues act as conformational randomizers close to the enthalpy-entropy compensation temperature, eliminating intrinsic biases in favor of largely balanced pPII/β dominated ensembles at physiological temperatures.

Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite of extensive efforts, structural data for PR photointermediate states have not been obtained. Based on DNP-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating 13C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the de-protonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signalling cascades.

Short peptides have been shown to be model systems for obtaining the conformational propensities of individual amino acids. Recent studies on cationic GxG (x: different guest amino acid residues) peptides in water revealed the Ramachandran plots for 16 amino acid residues. Out of the natural amino acids, alanine in GAG has been shown to stand out owing to its unusually high propensity for polyproline II (pPII) conformations (0.72). Thus, its conformational distribution differs substantially from the classical ones of Ramachandran and Flory. Peptide-water interactions have been proposed as the major determinant of alanine's preference for pPII. To elucidate how peptide hydration affects conformational preferences of alanine we used FTIR, vibrational circular dichroism, polarized Raman and NMR spectroscopy to determine Ramachandran plots of GAG in aqueous solutions with 3, 12, and 42mol% ethanol. We observed a modest decrease of the pPII fraction from 0.72 to 0.65 and an increase in β-strand population from 0.15 to 0.20 from pure water to 12 mol% ethanol. Interestingly, the corresponding changes of the enthalpic and entropic differences between pPII and β -strand by far exceed changes in Gibbs energy at room temperature owing to enthalpy-entropy compensation effects. Our thermodynamic data reveal that the cosolvent stabilizes pPII enthalpically (−10 kJ/mol in water to −18 kJ/mol in 12 mol% ethanol) but destabilizes it even more entropically (−23 to −50 J/mol∗K). Comparisons of IR and NMR spectra of GAG and N- methylacetamide in the same ethanol/water mixtures reveal changes of peptide hydration which causes the observed destabilization of pPII of alanine and the concomitant increase of its conformational entropy.