ABSTRACT
Human telomeres (HTs) can form DNA G-quadruplex (G4), an attractive target for anticancer and antiviral drugs. HT-G4s exhibit inherent structural polymorphism, posing challenges for understanding their specific recognition by ligands. Here, we aim to explore the impact of different topologies within a small segment of the HT (Tel22) on its interaction with BRACO19, a rationally designed G4 ligand with high quadruplex affinity, already employed in in-vivo treatments. Our multi-technique approach is based on the combined use of a set of contactless spectroscopic tools. Circular dichroism and UV resonance Raman spectroscopy probe ligand-induced conformational changes in the G4 sequence, while UV-visible absorption, coupled with steady-state fluorescence spectroscopy, provides further insights into the electronic features of the complex, exploiting the photoresponsive properties of BRACO19. Overall, we find that modifying the topology of the unbound Tel22 through cations (K+ or Na+), serves as a critical determinant for ligand interactions and binding modes, thus influencing the HT-G4's assembly capabilities. Furthermore, we show how fluorescence serves as a valuable probe for recognizing cation-driven multimeric structures, which may be present in living organisms, giving rise to pathological forms.
ABSTRACT
Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.
Subject(s)
Bile Pigments , Photoreceptors, Microbial , Photochemistry , Biliverdine , Bacterial Proteins/metabolism , Photoreceptors, Microbial/chemistry , LightABSTRACT
High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.
Subject(s)
Artifacts , Lasers , Myoglobin , Crystallography/instrumentation , Crystallography/methods , Electrons , Myoglobin/chemistry , Myoglobin/metabolism , Myoglobin/radiation effects , Photons , Protein Conformation/radiation effects , Quantum Theory , X-RaysABSTRACT
Relevant events during protein function such as ligand binding/release and interaction with substrates or with light are often accompanied by out-of-equilibrium structural dynamics. Time-resolved experimental techniques have been developed to follow protein structural changes as they happen in real time after a given reaction-triggering event. Time-resolved X-ray solution scattering is a promising approach that bears structural sensitivity with temporal resolution in the femto-to-millisecond time range, depending on the X-ray source characteristics and the triggering method. Here we present the basic principles of the technique together with a description of the most relevant results recently published and a discussion on the computational methods currently developed to achieve a structural interpretation of the time-resolved X-ray solution scattering experimental data.
Subject(s)
Proteins , X-Rays , X-Ray Diffraction , Scattering, Small Angle , Proteins/chemistryABSTRACT
CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.
Subject(s)
Cold Temperature , Histidine , Ligands , Oxidation-Reduction , LightingABSTRACT
G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions, G4s undergo multimerization. Here, we investigate the stacking interactions and structural features of telomeric G4 multimers by means of a novel low-resolution structural approach that combines small-angle X-ray scattering (SAXS) with extremely coarse-grained (ECG) simulations. The degree of multimerization and the strength of the stacking interaction are quantitatively determined in G4 self-assembled multimers. We show that self-assembly induces a significant polydispersity of the G4 multimers with an exponential distribution of contour lengths, consistent with a step-growth polymerization. On increasing DNA concentration, the strength of the stacking interaction between G4 monomers increases, as well as the average number of units in the aggregates. We utilized the same approach to explore the conformational flexibility of a model single-stranded long telomeric sequence. Our findings indicate that its G4 units frequently adopt a beads-on-a-string configuration. We also observe that the interaction between G4 units can be significantly affected by complexation with benchmark ligands. The proposed methodology, which identifies the determinants that govern the formation and structural flexibility of G4 multimers, may be an affordable tool aiding in the selection and design of drugs that target G4s under physiological conditions.
Subject(s)
DNA , G-Quadruplexes , Humans , Scattering, Small Angle , X-Ray Diffraction , DNA/chemistry , TelomereABSTRACT
Guanine-rich DNA sequences can fold into non-canonical nucleic acid structures called G-quadruplexes (G4s). These nanostructures have strong implications in many fields, from medical science to bottom-up nanotechnologies. As a result, ligands interacting with G4s have attracted great attention as candidates in medical therapies, molecular probe applications, and biosensing. In recent years, the use of G4-ligand complexes as photopharmacological targets has shown significant promise for developing novel therapeutic strategies and nanodevices. Here, we studied the possibility of manipulating the secondary structure of a human telomeric G4 sequence through the interaction with two photosensitive ligands, DTE and TMPyP4, whose response to visible light is different. The effect of these two ligands on G4 thermal unfolding was also considered, revealing the occurrence of peculiar multi-step melting pathways and the different attitudes of the two molecules on the quadruplex stabilization.
Subject(s)
G-Quadruplexes , Humans , Ligands , Light , Telomere/geneticsABSTRACT
Taking into account the presence of the crowded environment of a macromolecule has been an important goal of biology over the past 20 years. Molecular crowding affects the motions, stability and the kinetic behaviour of proteins. New powerful approaches have recently been developed to study molecular crowding, some of which make use of the synchrotron radiation light. The meeting "New Frontiers in Molecular Crowding" was organized in July 2022at the European Synchrotron Radiation facility of Grenoble to discuss the new frontiers of molecular crowding. The workshop brought together researchers from different disciplines to highlight the new developments of the field, including areas where new techniques allow the scientists to gain unprecedently expected information. A key conclusion of the meeting was the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives that could let the molecular crowding field flourish further.
ABSTRACT
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined inâ vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both inâ vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.
Subject(s)
Escherichia coli , Microscopy , Escherichia coli/metabolism , Green Fluorescent Proteins , Luminescent Proteins/chemistryABSTRACT
Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.
Subject(s)
Bacillus thuringiensis , Nanoparticles , Animals , Bacterial Proteins/toxicity , Endotoxins , Hemolysin Proteins/toxicity , Larva , Mosquito ControlABSTRACT
The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 µs after absorption of a first photon.
Subject(s)
Bacterial Proteins , Cyanobacteria , Bacterial Proteins/metabolism , Carotenoids/metabolismABSTRACT
Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state. Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer. The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into µm sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.
Subject(s)
Neutrons , Water , Neutron Diffraction/methods , Powders/chemistry , Proteins , Spectrum Analysis , Water/chemistryABSTRACT
Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light-activated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time-resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods.
Subject(s)
Crystallography, X-Ray , Protein Conformation/radiation effects , Proteins/ultrastructure , Lasers , Light , Models, Molecular , Proteins/chemistry , Proteins/radiation effects , Time Factors , X-Ray DiffractionABSTRACT
Nucleic acid sequences rich in guanines can organize into noncanonical DNA G-quadruplexes (G4s) of variable size. The design of small molecules stabilizing the structure of G4s is a rapidly growing area for the development of novel anticancer therapeutic strategies and bottom-up nanotechnologies. Among a multitude of binders, porphyrins are very attractive due to their light activation that can make them valuable conformational regulators of G4s. Here, a structure-based strategy, integrating complementary probes, is employed to study the interaction between TMPyP4 porphyrin and a 22-base human telomeric sequence (Tel22) before and after irradiation with blue light. Porphyrin binding is discovered to promote Tel22 dimerization, while light irradiation of the Tel22-TMPyP4 complex controls dimer fraction. Such a change in quaternary structure is found to be strictly correlated with modifications at the secondary structure level, thus providing an unprecedented link between the degree of dimerization and the underlying conformational changes in G4s.
Subject(s)
DNA/chemistry , G-Quadruplexes , Porphyrins/chemistry , X-Rays , Circular Dichroism , Dimerization , Molecular Structure , Scattering, Radiation , TelomereABSTRACT
The interaction between proteins and hydration water stabilizes protein structure and promotes functional dynamics, with water translational motions enabling protein flexibility. Engineered solvent-free protein-polymer hybrids have been shown to preserve protein structure, function, and dynamics. Here, we used neutron scattering, protein and polymer perdeuteration, and molecular dynamics simulations to explore how a polymer dynamically replaces water. Even though relaxation rates and vibrational properties are strongly modified in polymer coated compared to hydrated proteins, liquidlike polymer dynamics appear to plasticize the conjugated protein in a qualitatively similar way as do hydration-water translational motions.
Subject(s)
Polymers/chemistry , Proteins/chemistry , Diamines/chemistry , Glycolates/chemistry , Hydrogen Bonding , Molecular Dynamics Simulation , Myoglobin/chemistry , Neutron Diffraction , Polyethylene Glycols/chemistry , Protein Conformation , Thermodynamics , Water/chemistryABSTRACT
Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment, and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here, we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, in which the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced x-ray emission and x-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.
Subject(s)
Amyloid , Zinc , Humans , Insulin , Kinetics , X-Ray Absorption SpectroscopyABSTRACT
Proteins can misfold and form either amorphous or organized aggregates with different morphologies and features. Aggregates of amyloid nature are pathological hallmarks in so-called protein conformational diseases, including Alzheimer's and Parkinson's. Evidence prevails that the transient early phases of the reaction determine the aggregate morphology and toxicity. As a consequence, real-time monitoring of protein aggregation is of utmost importance. Here, we employed time-resolved neutron backscattering spectroscopy to follow center-of-mass self-diffusion and nano- to picosecond internal dynamics of lysozyme during aggregation into a specific ß-sheet rich superstructure, called particulates, formed at the isoelectric point of the protein. Particulate formation is found to be a one-step process, and protein internal dynamics, to remain unchanged during the entire aggregation process. The time-resolved neutron backscattering spectroscopy approach developed here, in combination with standard kinetics assays, provides a unifying framework in which dynamics and conformational transitions can be related to the different aggregation pathways.
Subject(s)
Muramidase/chemistry , Protein Aggregates , Spectrum Analysis/methods , Diffusion , Kinetics , Models, Molecular , Neutrons , Protein ConformationABSTRACT
Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
ABSTRACT
Proteins are the molecular workhorses in a living organism. Their 3D structures are animated by a multitude of equilibrium fluctuations and specific out-of-equilibrium motions that are required for proteins to be biologically active. When studied as a function of temperature, functionally relevant dynamics are observed at and above the so-called protein dynamical transition (~240 K) in hydrated, but not in dry proteins. In this review we present and discuss the main experimental and computational results that provided evidence for the dynamical transition, with a focus on the role of hydration water dynamics in sustaining functional protein dynamics. The coupling and mutual influence of hydration water dynamics and protein dynamics are discussed and the hypotheses illustrated that have been put forward to explain the physical origin of their onsets.
Subject(s)
Proteins/chemistry , Water/chemistry , TemperatureABSTRACT
Phytochromes are photoreceptor proteins that transmit a light signal from a photosensory region to an output domain. Photoconversion involves protein conformational changes whose nature is not fully understood. Here, we use time-resolved X-ray scattering and optical spectroscopy to study the kinetics of structural changes in a full-length cyanobacterial phytochrome and in a truncated form with no output domain. X-ray and spectroscopic signals on the µs/ms timescale are largely independent of the presence of the output domain. On longer time-scales, large differences between the full-length and truncated proteins indicate the timeframe during which the structural transition is transmitted from the photosensory region to the output domain and represent a large quaternary motion. The suggested independence of the photosensory-region dynamics on the µs/ms timescale defines a time window in which the photoreaction can be characterized (e.g. for optogenetic design) independently of the nature of the engineered output domain.