Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA-protein coupling.

Funder: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research); doi: https://doi.org/10.13039/501100003246Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein-DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes

Combined docking and machine learning identify key molecular determinants of ligand pharmacological activity on β2 adrenoceptor

G protein-coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands that bind them. Ligands exert their action via the interactions in the ligand binding pocket. We hypothesized that there is a common set of receptor interactions made by ligands of diverse structures that mediate their action and that among a large dataset of different ligands, the functionally important interactions will be over-represented. We computationally docked ~2700 known β2AR ligands to multiple β2AR structures, generating ca 75 000 docking poses and predicted all atomic interactions between the receptor and the ligand. We used machine learning (ML) techniques to identify specific interactions that correlate with the agonist or antagonist activity of these ligands. We demonstrate with the application of ML methods that it is possible to identify the key interactions associated with agonism or antagonism of ligands. The most representative interactions for agonist ligands involve K972.68×67 , F194ECL2 , S2035.42×43 , S2045.43×44 , S2075.46×641 , H2966.58×58 , and K3057.32×31 . Meanwhile, the antagonist ligands made interactions with W2866.48×48 and Y3167.43×42 , both residues considered to be important in GPCR activation. The interpretation of ML analysis in human understandable form allowed us to construct an exquisitely detailed structure-activity relationship that identifies small changes to the ligands that invert their pharmacological activity and thus helps to guide the drug discovery process. This approach can be readily applied to any drug target

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs

Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling

Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein–DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes.BN/Sander Tans La

Structural characterization of ¹³C-enriched humins and alkali-treated ¹³C humins by 2D solid-state NMR

Humin by-products are formed during the acid-catalyzed dehydration of carbohydrates to bio-based platform molecules, such as hydroxymethylfurfural and levulinic acid. The molecular structure of these humins has not yet been unequivocally established. 1D 13C solid-state NMR data reported have, for example, provided considerable insight, but do not allow for the unambiguous assignment of key structural motifs. Complementary (2D) techniques are needed to gain additional insight into the molecular structure of humins. Here, the preparation of 13C-enriched humins is reported, together with the reactive solubilization of these labeled humins and their characterization with complementary 1D and 2D solid-state NMR techniques. 1D cross polarization (CP) and direct excitation (DE) 13C solid-state NMR spectra, 2D 13C-detected double-quantum single-quantum (DQSQ) as well as 2D 1H-detected heteronuclear correlation (HETCOR) were recorded with different excitation schemes. These experiments unambiguously established that the original humins have a furan-rich structure with aliphatic linkers and allowed for a refinement of the molecular structure proposed previously. Solid-state NMR data of alkali-treated 13C-labeled humins showed that an arene-rich structure is formed at the expense of the furanic network during alkaline pretreatment

Biomolecular DNP-Supported NMR Spectroscopy using Site-Directed Spin Labeling

International audienceDynamic nuclear polarization (DNP) has beenshown to greatly enhance spectroscopic sensitivity, creatingnovel opportunities for NMR studies on complex and largemolecular assemblies in life and material sciences. In suchapplications, however, site-specificity and spectroscopic resolutionbecome critical factors that are usually difficult to controlby current DNP-based approaches. We have examinedin detail the effect of directly attaching mono- or biradicalsto induce local paramagnetic relaxation effects and, at thesame time, to produce sizable DNP enhancements. Usinga membrane-embedded ion channel as an example, wevaried the degree of paramagnetic labeling and the locationof the DNP probes. Our results show that the creation oflocal spin clusters can generate sizable DNP enhancementswhile preserving the intrinsic benefits of paramagnetic relaxationenhancement (PRE)-based NMR approaches. DNPusing chemical labeling may hence provide an attractiveroute to introduce molecular specificity into DNP studies inlife science applications and beyond

Direct observation of Hsp90-induced compaction in a protein chain

The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70.BN/Sander Tans La

Protein chain collapse modulation and folding stimulation by GroEL-ES

The collapse of polypeptides is thought important to protein folding, aggregation, intrinsic disorder, and phase separation. However, whether polypeptide collapse is modulated in cells to control protein states is unclear. Here, using integrated protein manipulation and imaging, we show that the chaperonin GroEL-ES can accelerate the folding of proteins by strengthening their collapse. GroEL induces contractile forces in substrate chains, which draws them into the cavity and triggers a general compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL and is aided by GroES binding to the cavity rim and by the amphiphilic C-terminal tails at the cavity bottom. Collapse modulation is distinct from other proposed GroEL-ES folding acceleration mechanisms, including steric confinement and misfold unfolding. Given the prevalence of collapse throughout the proteome, we conjecture that collapse modulation is more generally relevant within the protein quality control machinery. BN/Sander Tans La

NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field

Dynamic nuclear polarization (DNP) has become a powerful method to enhance spectroscopic sensitivity in the context of magnetic resonance imaging and nuclear magnetic resonance spectroscopy. We show that, compared to DNP at lower field (400 MHz/263 GHz), high field DNP (800 MHz/527 GHz) can significantly enhance spectral resolution and allows exploitation of the paramagnetic relaxation properties of DNP polarizing agents as direct structural probes under magic angle spinning conditions. Applied to a membrane-embedded K+ channel, this approach allowed us to refine the membrane-embedded channel structure and revealed conformational substates that are present during two different stages of the channel gating cycle. High-field DNP thus offers atomic insight into the role of molecular plasticity during the course of biomolecular function in a complex cellular environment