Intramolecular Conformational Dynamics Research Articles

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Ensemble Switching of the DNA-Binding Domain of Human FoxP1

The P-subfamily of Forkhead box (FoxP) transcription factors (TFs) are crucial to the normal development of the central nervous system. Over 25 mutations link the FoxP family with neurodevelopmental disorders (NDD), including Autism Spectrum Disorder, mild-to-moderate intellectual disabilities, obsessions and compulsions, sensory integration disorder, aggression, hyperactivity, and moderate-to-severe expressive and receptive language impairments. We have previously shown that intrinsically disordered regions of the DNA-binding domain of FoxP1 are observed during domain swapping homodimerization. However, we know little about the structural arrangement before the formation of the dimer. Therefore, we use single-molecule (sm) Förster Resonance Energy Transfer (FRET) and molecular dynamics simulations to evaluate the intramolecular FoxP1 conformational space that enables domain swapping. Moreover, we monitor the impact that DNA has in the monomeric and dimeric conditions by monitoring the intramolecular conformational dynamics using a series of double FRET labeled variants. Our data show that the monomeric FoxP1 samples a heterogeneous conformational ensemble, switching between a folded and an expanded ensemble. We corroborate these observations by replica-exchange Discrete Molecular Dynamics (rxDMD) simulations and identify locally disordered regions responsible for the ensemble switching. Finally, we show that DNA stabilizes the folded conformation in the monomer and dimer species whereas the intramolecular flexibility of FoxP1 decreases mainly in dimeric conditions. in summary, we propose that FoxP1 evolved to dimerize and bind as a dimer to DNA due to the dimer’s stabilizing effect. Funding: FONDECYT 1170701; NIH 1P20GM12134201; NSF CAREER MCB-1749778; ANID-Millennium Science initiative Program-ICN17_022.

Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS.

Fӧrster-type resonance energy transfer (FRET) with fluorescence cross-correlation spectroscopy (FCCS) is a powerful combination for observing intramolecular conformational dynamics on the micro- to millisecond timescale. Owing to its sensitivity to various physical parameters, FRET-FCCS has also been used to detect the reagent effects on proteins dynamics. However, FRET-FCCS alone cannot acquire the exact measurements of rate constants. Moreover, this technique is highly model dependent and can be unreliable when determining too many parameters at once. On the contrary, single-molecular FRET (smFRET) can measure the conformational states and their populations directly, although it is extremely challenging for probing fast dynamics under 1ms. In this chapter, we describe how to realize sub-millisecond conformational dynamics measurements of a SNARE protein Ykt6 under lipid environments by smFRET and FRET-FCCS. This protocol includes sample preparation, microscope designs, data acquisition, and analysis methodology.

Intermolecular correlations are necessary to explain diffuse scattering from protein crystals.

Conformational changes drive protein function, including catalysis, allostery and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work has challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid-body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signal reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid-body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.

Synthesis and Direct Observation of Thermoresponsive DNA Copolymers.

Single-molecule techniques allow for the direct observation of long-chain macromolecules, and these methods can provide a molecular understanding of chemically heterogeneous and stimuli-response polymers. In this work, we report the synthesis and direct observation of thermoresponsive DNA copolymers using single-molecule techniques. DNA-PNIPAM copolymers are synthesized using a two-step strategy based on polymerase chain reaction (PCR) for generating linear DNA backbones containing non-natural nucleotides (dibenzocyclooctyne-dUTP), followed by grafting thermoresponsive side branches (poly(N-isopropylacrylamide), PNIPAM) onto DNA backbones using copper-free click chemistry. Single-molecule fluorescence microscopy is used to directly observe the stretching and relaxation dynamics of DNA-PNIPAM copolymers both below and above the lower critical solution temperature (LCST) of PNIPAM. Our results show that the intramolecular conformational dynamics of DNA-PNIPAM copolymers are affected by temperature, branch density, and branch molecular weight. Single-molecule experiments reveal an underlying molecular heterogeneity associated with polymer stretching and relaxation behavior, which arises in part due to heterogeneous chemical identity on DNA copolymer dynamics.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Forster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6.

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding

The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1–88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22–33 and in the Myc homology box I (MBI; residues 45–65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1–88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

Detecting Intramolecular Conformational Dynamics of Single Molecules in Short Distance Range with Subnanometer Sensitivity

Single molecule detection is useful for characterizing nanoscale objects such as biological macromolecules, nanoparticles and nanodevices with nanometer spatial resolution. Fluorescence resonance energy transfer (FRET) is widely used as a single-molecule assay to monitor intramolecular dynamics in the distance range of 3-8 nm. Here we demonstrate that self-quenching of two rhodamine derivatives can be used to detect small conformational dynamics corresponding to subnanometer distance changes in a FRET-insensitive short-range at the single molecule level. A ParM protein mutant labeled with two rhodamines works as a single molecule adenosine 5'-diphosphate (ADP) sensor that has 20 times brighter fluorescence signal in the ADP bound state than the unbound state. Single molecule time trajectories show discrete transitions between fluorescence on and off states that can be directly ascribed to ADP binding and dissociation events. The conformational changes observed with 20:1 contrast are only 0.5 nm in magnitude and are between crystallographic distances of 1.6 and 2.1 nm, demonstrating exquisite sensitivity to short distance scale changes. The systems also allowed us to gain information on the photophysics of self-quenching induced by rhodamine stacking: (1) photobleaching of either of the two rhodamines eliminates quenching of the other rhodamine fluorophore and (2) photobleaching from the highly quenched, stacked state is only 2-fold slower than from the unstacked state.

Restricted conformation dynamics of single functionalized perylene bisimide molecules on SiO2 surfaces and in thin polymer films

Intramolecular conformational dynamics caused by the bay groups of perylene bisimide (PBI) molecules have been investigated by single molecule spectroscopy in thin PMMA polymer films and on SiO2 surfaces. All dynamic processes occur in a jump-like fashion on SiO2 but in a likewise continuous way in PMMA. Surface attachment on SiO2 is accomplished by pyridyl functionalization of PBI demonstrating a nearly perpendicular orientation of the long axis of perylene as has been concluded from polarization experiments. This orientation is subject to fluctuations of the molecular orientation and/or conformation, which are considerably reduced in PMMA. According to static and time-dependent fluorescence spectra at least 2 different conformations have been identified.

Active Intramolecular Conformational Dynamics Controlling the Assembly of Azobenzene Derivatives at Surfaces

Molecular self-assembly at surfaces proceeds through complex selective pathways involving not only the recognition of molecular structures based on lock-and-key interactions, but also the existence of effective methods of dynamical search and trial. This becomes especially crucial when a mixture of different molecules with immiscible shapes results in the segregation of mesoscopic separated phases. For example, this is the case in the formation of large homochiral phases from a racemic mixture of chiral molecules at surfaces. Large-scale mass transport through thermally activated diffusion has been proposed as the mechanism leading to phase segregation. However, it is not clear how molecules of one species can move for large distances, in many cases overcoming barriers associated with topological defects such as monoatomic steps. In solution, the conformational dynamics of molecules with internal flexibility plays an important role in supramolecular assembly. For example, the folding structure of proteins or peptides is governed by dynamical stochastic mapping of multidimensional conformational configurations. A metal surface represents in turn a rigid object interacting with the molecular adsorbates, in many cases very strongly compared to the magnitude of thermal energy at room temperature. In spite of that, thermally activated conformational changes have been identified at the single-molecule scale, which implies that internal degrees of freedom may also be active during the recognition and growth of molecular thin films at surfaces. Whether they play a crucial role in the mesoscopic ordering and phase separation is not yet clear. Here, we use low-temperature (5 K) scanning tunneling microscopy (STM) to statistically track the different molecular structures of an azobenzene derivative adsorbed on a gold surface. We find that the ratio between different azobenzene rotamers changes in response to an increase in temperature and coverage, thus proving that intramolecular conformational dynamics is an active pathway of molecular recognition during the formation of large-scale molecular assemblies on surfaces. Our study is performed on 3,3’-di(methoxycarbonyl)azobenzene (CMA) deposited on a Au ACHTUNGTRENNUNG(111) surface. A gold substrate is employed because it leads to weak molecular adsorption, thus maximizing the role of intermolecular interactions. CMA is formed by an azobenzene skeleton functionalized with a carboxymethyl end group at one of the meta sites of the phenyl rings (see Figures 1a and b). The methyl part of the end group

Near-Field Scanning Optical Microscopy of Single Fluorescent Dendritic Molecules

Individual dendritic molecules adsorbed o­n glass containing a single fluorescent rhodamine B core have been observed with near-field scanning optical microscopy (NSOM); height and fluorescence images were obtained simultaneously. The dendritic assemblies can be discriminated from free fluorescent cores o­n the basis of accurate simultaneous localization of both the fluorescent core and the surrounding dendritic shell. There are no significant differences between the photophysical properties of the free and dendritic fluorophores. The full three- dimensional orientation of each individual fluorescent core can be resolved and millisecond time resolution accuracy has been achieved. Most dendritic structures exhibited rotational motion of the fluorescent core o­n a millisecond-second time scale, revealing intramolecular conformational dynamics

Analytical ultracentrifugation of chromatin.

The ability of analytical ultracentrifugation to elucidate chromatin structure/function relationships originates directly from its capacity to accurately measure key structural properties of complex macromolecular assemblies in solution. Figure 1 schematically illustrates the complex nature of chromatin. Newly replicated DNA is wrapped around core histone octamers spaced at approx 200 bp intervals to form nucleosomal arrays, which then interact with linker histones and numerous other nonhistone chromosomal proteins to form “chromatin.” Chromatin is conformationally dynamic, undergoing a number of short-range and long-range folding transitions to produce highly condensed interphase chromosomal fibers (Fig. 1). For short chromatin fragments studied in vitro, fiber condensation manifests both in the form of intramolecular conformational changes and reversible oligomerization (, , , ). In addition, the structure of chromatin fibers and functions such as transcription and replication are irrevocably linked; any given region of a chromosomal fiber can be either functionally active or inactive depending on both its specific complement of chromatin-associated proteins and its overall state of condensation (,). Consequently, to biochemically characterize chromatin structure/function relationships in vitro, one must be able to analyze both the intramolecular conformational dynamics and intermolecular interactions of an exceedingly complex macromolecular assembly (e.g., a 12-mer nucleosomal array containing one H1 molecule per nucleosome consists of >100 proteins and 2400 bp of DNA, has a molecular mass of approx 3.5×106 D, yet represents only roughly one millionth of an intact eukaryotic chromosome.) Open image in new window Fig. 1. Schematic illustration of the hierarchical relationships between DNA, chromatin, and interphase chromosomal fibers.