Fluorescence Resonance Energy Transfer Spectroscopy Research Articles

Mei5-Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA.

Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5-Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5-Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5-Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5-Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5-Sae3 stabilized Dmc1 nucleating clusters with two to threemolecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5-Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5-Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.

Spectroscopic and computational studies on the binding interaction of biologically active thioridazine and perphenazine with human Matrix metalloproteinases 9

Matrix metaloproteinase (MMP)-9, as a neuroinflammation-facilitating element, plays a significant role in neurodegenerative disorders and could be considered an inflammation control target. This study is intended to survey the effects of thioridazine (TRZ) and phenothiazines (PRZ) as neuroleptic drugs on the performance of recombinant human MMP-9 (rhMMP-9) by using experimental (ultraviolet visible (UV–vis), fluorescence, synchronous fluorescence, fluorescence resonance energy transfer (FRET) spectroscopy, circular dichroism (CD), surface plasmon resonance (SPR), and theoretical techniques (molecular docking). For this purpose, rhMMP-9 was expressed and purified by utilizing recombinant E. coli bacteria. The kinetic analysis illustrated that the different concentrations of TRZ and PRZ could considerably decrease the catalytic function of rhMMP-9 through competitive and mixed inhibition mechanisms, respectively. The results illustrated the higher affinity of TRZ-MMP-9 with an inhibitory IC50 value of 10.7 µM in comparison with PRZ (12.1 µM) following the conformational alteration of the enzyme. In addition, hydrophobic binding and hydrogen bonds/van der Waals forces were suggested for TRZ-MMP-9 and PRZ-MMP-9, respectively. Finally, the molecular docking study demonstrated that TRZ and PRZ bind to the cavity in the catalytic and fibronectin domains, respectively, which supports the experimental data. This study provides some experimental and structural insights into the possible anti-inflammatory mechanisms of TRZ and PRZ drugs that suggest both drugs could be used as anti-MMP-9 drugs in neuroinflammatory conditions.

Linear spectral unmixing analysis in single-molecule FRET spectroscopy for fluorophores with large spectral overlap.

Fluorescence resonance energy transfer (FRET) is a highly useful tool to investigate biomolecular interactions and dynamics in single-molecule spectroscopy and nanoscopy. However, the use of spectrally overlapping dye pairs results in various artifact signals that prevent accurate determination of FRET values. In this paper, an algorithmic method of spectral unmixing was devised to extract FRET values of spectrally overlapping dye pairs at the single molecule level. Application of this method allows the determination of both the donor-acceptor composition and the FRET efficiency of the samples labelled with spectrally overlapping dye pairs.

Structural Dynamics Role of AGG Interruptions in Inhibition CGG Repeat Expansion Associated with Fragile X Syndrome.

Abnormal expansion of trinucleotide CGG repeats is responsible for Fragile X syndrome. AGG interruptions in CGG repeat tracts were found in most healthy individuals, suggesting a crucial role in preventing disease-prone repeat expansion. Previous biophysics studies emphasize a difference in the secondary structure affected by AGG interruptions. However, the mechanism of how AGG interruptions impede repeat expansion remains elusive. We utilized single-molecule fluorescence resonance energy transfer spectroscopy to investigate the structural dynamics of CGG repeats and their AGG-interrupted variants. Tandem CGG repeats fold into a stem-loop hairpin structure with the capability to undergo a conformational rearrangement to modulate the length of the overhang. However, this conformational rearrangement is much more retarded when two AGG interruptions are present. Considering the significance of hairpin slippage in repeat expansion, we present a molecular basis suggesting that the internal loop created by two AGG interruptions acts as a barrier, obstructing the hairpin slippage reconfiguration. This impediment potentially plays a crucial role in curbing abnormal expansion, thereby contributing to the genomic stability.

Protein Labeling Facilitates the Understanding of Protein Corona Formation via Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy.

Once nanoparticles enter into the biological milieu, nanoparticle-biomacromolecule complexes, especially the protein corona, swiftly form, which cause obvious effects on the physicochemical properties of both nanoparticles and proteins. Here, the thermodynamic parameters of the interactions between water-soluble GSH-CdSe/ZnS core/shell quantum dots (GSH-QDs) and human serum albumin (HSA) were investigated with the aid of labeling fluorescence of HSA. It was proved that the labeling fluorescence originating from a fluorophore (BDP-CN for instance) could be used to investigate the interactions between QDs and HSA. Gel electrophoresis displayed that the binding ratio between HSA and QDs was ∼2:1 by direct visualization. Fluorescence resonance energy transfer (FRET) results indicated that the distance between the QDs and the fluorophore BDP-CN in HSA was 7.2 nm, which indicated that the distance from the fluorophore to the surface of the QDs was ∼4.8 nm. Fluorescence correlation spectroscopy (FCS) results showed that HSA formed a monolayer of a protein corona with a thickness of 5.5 nm. According to the spatial structure of HSA, we could speculate that the binding site of QDs was located at the side edge (not the triangular plane) of HSA with an equilateral triangular prism. The elaboration of the thermodynamic parameters, binding ratio, and interaction orientation will highly improve the fundamental understanding of the formation of protein corona. This work has guiding significance for the exploration of the interactions between proteins and nanomaterials.

Nucleotide dependent conformational changes of MutL.

MutL is one of the enzymes required for mismatch repair (MMR). Mutations in MutL are linked to Lynch Syndrome, which predisposes individuals to colorectal and other cancers. In MMR, MutL is known to nick the error-containing daughter strand of mismatch DNA as well as form complexes with other MMR proteins such as MutS. MutL accomplishes this by undergoing a series of conformational changes which include extended, one-arm, semi-condensed, and condensed states in the presence of ATP. These dynamic conformational changes are needed to coordinate MMR, but the exact sequence of events and the impact of disease-associated mutations are unknown. We used site-directed mutagenesis to fluorescently label sites in the different domains of Thermus Aquaticus MutL, including the N-terminal, linker arms, and C-terminal domains. We expressed MutL variants with wild-type and ATPase-deficient activity and monitored their dynamic conformational changes under differing nucleotide conditions with pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) in solutions of freely diffusing molecules. Using the same technique, we also monitored the interactions of MutL with MutS, the enzyme that initially recognizes a mismatch, to reveal the specific interactions and conformational changes that are necessary to initiate mismatch repair.

An integrative approach to molecular dynamics and single molecule FRET techniques.

Molecular dynamics (MD) simulation and single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy are two techniques that can be used to study conformational heterogeneity of proteins. However, significant challenges exist in these techniques that limit their effectiveness. All-atom MD is currently too slow to adequately sample conformational space of proteins. From an experimental perspective, deciding where to place the fluorescent dye reporters for smFRET measurements can lead to a great deal of wasted time, effort, and expense in producing labeled proteins that may or may not provide useful information. Even if the dyes are suitably placed, interpretation of smFRET data is also challenging since many conformations can lead to the same FRET efficiency for a single smFRET-labeled protein. In this work, we have addressed these problems using a highly integrative approach to smFRET and MD. We have developed an iterative method to characterize the conformational ensemble of proteins by combining MD-based enhanced sampling techniques and smFRET spectroscopy, where the computational and experimental approaches can benefit mutually from each other. We first employed an enhanced sampling based MD method (ES-MD) using system-specific collective variables for the system under study to sample various conformations. Since these simulations are biased, the relative weight of different populations of samples may not be accurate. In the next step, we predicted the FRET efficiency distributions for a large number of residue pairs and from the imperfect results determined the residues that are predicted to provide the most informative FRET distributions. The measured smFRET data was then used within a resampling scheme to correct the original computed set of conformations, which was then used to predict additional smFRET distributions for other labeled residues. We have examined this methodology on intrinsically-disordered/structured and membrane/soluble proteins.

Investigating the impact of ATPase mutation on MutL function.

DNA mismatch repair (MMR) is an enzymatic pathway for fixing incorrect base pairing that may arise during DNA replication. Mutations in the enzyme MutL have been linked to Lynch syndrome, which is the leading cause of hereditary colorectal cancer. MutL is an important part of the MMR pathway and is thought to nick the error containing DNA strand for repair as well as perform other critical functions. To accomplish this, MutL has several large conformational changes that occur in the presence of ATP. While we believe these conformational changes are important for MMR, the exact order of the process and the direct effect of disease-related mutations are unknown. To better understand this process, we used Thermus Aquaticus MutL as a model system for human MutLalpha and mutated the ATPase domain by changing the glutamic acid in residue 28 to an alanine (E28A, aligned to E34A in human MLH1, a common mutation in Lynch syndrome patients). We then conducted an ATPase assay to compare wild-type and E28A activity and fluorescently labeled each MutL variant for single molecule fluorescence studies. We monitored MutL conformational dynamics under varying nucleotide conditions using pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) to reveal the impact of the E28A mutation on the dynamic function of MutL.

Time-resolved mechanisms of essential biological pathways.

The diversity of enzyme structure and function in living systems leads to exciting opportunities in biophysical research. Large numbers of these protein machines coordinate across space and time and undergo drastic changes in shape to carry out complex biological processes. My lab focuses on investigating precisely how genetic mutations impact the molecular function of key enzymes involved in DNA repair and/or RNA processing pathways. We use single molecule time-resolved fluorescence spectroscopy as a tool to explore in real time the protein-nucleic acid interactions and dynamic structural rearrangements that drive specific pathways. We are currently studying the detailed mechanisms SARS-CoV-2 viral RNA processing, ribosome assembly, and DNA mismatch repair. I will present recent results from pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) experiments. We aim to completely characterize the impact of mutations linked to disease on the molecular function of enzymes to understand how complex diseases develop at the molecular level and to identify new therapeutic targets.

Rational design of a ratiometric fluorescent aptasensor for patulin in traditional Chinese medicine through the studies of the interaction mechanism between its DNA aptamer and the target molecule.

Patulin (PAT) is a kind of mycotoxin which must be monitored for the sake of quality and safety in traditional Chinese medicine (TCM) owing to its harm to human health. On this account, a rationally designed ratiometric fluorescent aptasensor was developed based on the studies of the interaction mechanism between PAT and its aptamer (PAT-APT). First, CD spectroscopy, molecular docking, and molecular dynamic simulation were applied to investigate the details on how PAT-APT binds with its target molecule. The results indicated that the structure of PAT-APT changed to a certain extent and was stabilized after binding with PAT. C-11, C-37 and C-38 were the key sites for the recognition and interaction between PAT-APT and its target. Second, based on these results, a ratiometric aptasensor was designed using fluorescence resonance energy transfer (FRET) and synchronous fluorescence spectroscopy. A complementary sequence (cDNA) to the aptamer with an appropriate length and hybridization position was obtained through rational design and optimization. Both PAT-APT and cDNA were labeled using a pair of fluorophores, which could generate FRET when the two single-stranded oligonucleotides hybridized. The accurate detection of PAT could be realized according to the change ratio of the fluorescence intensity at the corresponding wavelengths of the two fluorophores before and after the assay. The aptasensor achieved an ultralow limit of detection of 0.16 nM, perfect selectivity, and satisfactory practicability in complex TCM samples. To our knowledge, this is the first aptasensor for PAT designed through the interaction mechanism between its aptamer and the target molecule. Moreover, the assay for PAT is cost-effective, does not need complicated pretreatment and only takes less than an hour. In summary, this study makes a contribution to the safety control of TCM and provides a thinking mode from mechanism to rational design to conquer the problem of sensitive aptasensing of one component in a complex system.

Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity.

Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain's minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father's male development, but at odds with the daughter's XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably "unclamps" its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.

Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy.

Conformational dynamics in riboswitches involves ligand binding and folding of RNA, each of which can be influenced by excluded volume effects under "crowded" in vivo cellular conditions and thus incompletely characterized by in vitro studies under dilute buffer conditions. In this work, temperature-dependent single-molecule fluorescence resonance energy transfer (FRET) spectroscopy is used to characterize the thermodynamics of (i) cognate ligand and (ii) molecular crowders (PEG, polyethylene glycol) on folding of the B. subtilis LysC lysine riboswitch. With the help of detailed kinetic analysis, we isolate and study the effects of PEG on lysine binding and riboswitch folding steps individually, from which we find that PEG crowding facilitates riboswitch folding primarily via a surprising increase in affinity for the cognate ligand. This is furthermore confirmed by temperature-dependent studies, which reveal that PEG crowding is not purely entropic and instead significantly impacts both enthalpic and entropic contributions to the free energy landscape for folding. The results indicate that PEG molecular crowding/stabilization of the lysine riboswitch is more mechanistically complex and requires extension beyond the conventional picture of purely repulsive solvent-solute steric interactions arising from excluded volume and entropy. Instead, the current experimental FRET data support an alternative multistep mechanism, whereby PEG first entropically crowds the unfolded riboswitch into a "pre-folded" conformation, which in turn greatly increases the ligand binding affinity and thereby enhances the overall equilibrium for riboswitch folding.

Fluorescence Resonance Energy Transfer Measurements in Polymer Science: A Review.

Fluorescence resonance energy transfer (FRET) is a non-invasive characterization method for studying molecular structures and dynamics, providing high spatial resolution at nanometer scale. Over the past decades, FRET-based measurements are developed and widely implemented in synthetic polymer systems for understanding and detecting a variety of nanoscale phenomena, enabling significant advances in polymer science. In this review, the basic principles of fluorescence and FRET are briefly discussed. Several representative research areas are highlighted, where FRET spectroscopy and imaging can be employed to reveal polymer morphology and kinetics. These examples include understanding polymer micelle formation and stability, detecting guest molecule release from polymer host, characterizing supramolecular assembly, imaging composite interfaces, and determining polymer chain conformations and their diffusion kinetics. Finally, a perspective on the opportunities of FRET-based measurements is provided for further allowing their greater contributions in this exciting area.

Probing functional conformation-state fluctuation dynamics in recognition binding between calmodulin and target peptide.

Conformational dynamics play a crucial role in protein functions. A molecular-level understanding of the conformational transition dynamics of proteins is fundamental for studying protein functions. Here, we report a study of real-time conformational dynamic interaction between calcium-activated calmodulin (CaM) and C28W peptide using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy and imaging. Plasma membrane Ca-ATPase protein interacts with CaM by its peptide segment that contains 28 amino acids (C28W). The interaction between CaM and the Ca-ATPase is essential for cell signaling. However, details about its dynamic interaction are still not clear. In our current study, we used Cyanine3 labeled CaM (N-domain) and Dylight 649 labeled C28W peptide (N-domain) to study the conformational dynamics during their interaction. In this study, the FRET can be measured when the CaM-C28W complex is formed and only be observed when such a complex is formed. By using single-molecule FRET efficiency trajectory and unique statistical approaches, we were able to observe multiple binding steps with detailed dynamic features of loosely bound and tightly bound state fluctuations. The C-domain of CaM tends to bind with C28W first with a higher affinity, followed by the binding of the CaM N-domain. Due to the comparatively high flexibility and low affinity of the N-domain and the presence of multiple anchor hydrophobic residues on the peptide, the N-domain binding may switch between selective and non-selective binding states, while the C-domain remains strongly bound with C28W. The results provide a mechanistic understanding of the CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics.

Conformational states and fluctuations in endothelial nitric oxide synthase under calmodulin regulation

Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.

When two become one: Integrating FRET and EPR into one structural model

In modern structural biology, rarely one method can provide all structural insights, and integrative approaches combining different results into one framework are the necessary future. For example, cryo-electron microscopy/tomography and x-ray crystallography are well-established techniques yielding atomic-resolution structures of folded proteins. However, these types of structural information may be limited in providing insights on highly dynamic processes or intrinsically disordered proteins. Such data can be complemented by other classes of techniques, such as single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR) spectroscopy, which measure distances or even distance distributions between labels that can be site-specifically mounted into the biomolecule.

Fret Study of the Binding of HIV-1 RRE RNA to Rev Peptide

In the viral life cycle of HIV-1, binding of the Rev protein to the Rev Response Element (RRE) RNA to form the Rev-RRE complex is essential for viral RNA to be exported from the cell nucleus. This interaction has been suggested as a new therapeutic target, which makes an accurate and detailed understanding of Rev-RRE interactions essential. The m6A post-transcriptional modification is known to regulate gene expression and modulate infectivity of viral RNAs. The binding region of the RRE RNA has two known, conserved m6A methylation sites, but the effect and significance of these modifications is an active area of investigation. This study measures binding of an Alexa Fluor 594-labeled RNA hairpin that contains the high-affinity Rev-binding site to an Alexa Fluor 488-labeled peptide that contains the 17 amino acid binding region of the Rev protein as a model of the primary RRE-Rev binding site. Binding of Rev peptides to immobilized RNA hairpins is measured via single molecule fluorescence resonance energy transfer (FRET) spectroscopy. Single molecule measurements can access information hidden by averaging in bulk measurements, especially heterogeneities, such as multiple conformations, short lived intermediates, or varied kinetic rates. Statistical analysis and a kinetic model enable extraction of binding and dissociation rates for the Rev protein. The eventual goal of this study is a comparison of the binding rates for methylated and unmethylated variants of the RRE RNA, which will reveal whether m6A methylation significantly impacts the Rev-RRE binding rate at the primary binding site.

Affinity of Skp to OmpC revealed by single-molecule detection

Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

An Integrative Approach to Single-Molecule FRET Spectroscopy and Molecular Dynamics Simulations for the Study of Intrinsically Disordered Proteins

Recent technological advances in single-molecule techniques, such as single molecule fluorescence resonance energy transfer (smFRET) spectroscopy, have paved the way for the study of protein structural dynamics under biologically relevant conditions. Unlike high-resolution structure determination techniques such as x-ray crystallography, however, smFRET spectroscopy provides limited information spatially. Fortunately, the smFRET data combined with computational techniques such as molecular dynamics (MD) simulations could provide enough information to model the structural dynamics of protein at a spatiotemporal resolution. The starting point of these simulations is often based on crystal structure or other high-resolution structures of proteins. Unfortunately, such structures are not available for intrinsically disordered proteins (IDPs). Here we have employed enhanced sampling techniques in combination with all-atom MD and smFRET data to model the structural dynamics of IDPs. We have specifically employed state-of-the-art dimensionality reduction techniques in conjunction with enhanced sampling schemes to assimilate MD trajectories to make them consistent with the smFRET measurements. The novel methodology developed in this work uses the smFRET data within enhanced sampling techniques to first generate an ensemble of protein conformations and then predict FRET efficiency distributions for candidate labeled proteins. An iterative procedure is then used to efficiently design smFRET experiments based on the computational predictions and refine the conformational ensemble of protein, until a convergence is reached. The final step involves employing machine learning techniques to determine both the state distributions and their kinetics from smFRET-informed MD trajectories. We have successfully used this approach to study the structural dynamics of the C-terminal domain of membrane insertase Alb3, which is known to be intrinsically disordered. The novel approach developed here lays the groundwork for the efficient application of smFRET technique for the study of IDPs.

Periodic Operation of a Dynamic DNA Origami Structure Utilizing the Hydrophilic-Hydrophobic Phase-Transition of Stimulus-Sensitive Polypeptides.

Dynamic DNA nanodevices are designed to perform structure-encoded motion actuated by a variety of different physicochemical stimuli. In this context, hybrid devices utilizing other components than DNA have the potential to considerably expand the library of functionalities. Here, the reversible reconfiguration of a DNA origami structure using the stimulus sensitivity of elastin-like polypeptides is reported. To this end, a rectangular sheet made using the DNA origami technique is functionalized with these peptides and by applying changes in salt concentration the hydrophilic-hydrophobic phase transition of these peptides actuate the folding of the structure. The on-demand and reversible switching of the rectangle is driven by externally imposed temperature oscillations and appears at specific transition temperatures. Using transmission electron microscopy, it is shown that the structure exhibits distinct conformational states with different occupation probabilities, which are dependent on structure-intrinsic parameters such as the local number and the arrangement of the peptides on the rectangle. It is also shown through ensemble fluorescence resonance energy transfer spectroscopy that the transition temperature and thus the thermodynamics of the rectangle-peptide system depends on the stimuli salt concentration and temperature, as well as on the intrinsic parameters.