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.

Conformational dynamics of the hepatitis C virus 3'X RNA.

The 3' end of the hepatitis C virus genome is terminated by a highly conserved, 98 nt sequence called 3'X. This untranslated structural element is thought to regulate several essential RNA-dependent processes associated with infection. 3'X has two proposed conformations comprised of either three or two stem-loop structures that result from the different base-pairing interactions within the first 55 nt. Here, we used single-molecule Förster resonance energy transfer spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate this region of the RNA (3'X55). We observed that 3'X55 can adopt both proposed conformations and the relative abundance of them can be modulated by either solution conditions or nucleotide deletions. Furthermore, interconversion between the two conformations takes place over the course of several hours. The simultaneous existence of two slowly interconverting conformations may help prime individual copies of the viral genome for either viral protein or RNA synthesis, thereby minimizing conflicts between these two competing processes.

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.

Biophysical Investigation of RNA ⋅ DNA : DNA Triple Helix and RNA : DNA Heteroduplex Formation by the lncRNAs MEG3 and Fendrr.

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes.

Photobleaching FRET-FLIM-ICS for quaternary structure quantification on cells. Theory and simulations

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu.

A molecular dynamics simulation study of glycine/serine octapeptides labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene fluorophore.

The compound 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is a versatile fluorophore widely used in Förster resonance energy transfer (FRET) spectroscopy studies due to its remarkable sensitivity, enabling precise donor-acceptor distance measurements, even for short peptides. Integrating time-resolved and FRET spectroscopies with molecular dynamics simulations provides a robust approach to unravel the structure and dynamics of biopolymers in a solution. This study investigates the structural behavior of three octapeptide variants: Trp-(Gly-Ser)3-Dbo, Trp-(GlyGly)3-Dbo, and Trp-(SerSer)3-Dbo, where Dbo represents the DBO-containing modified aspartic acid, using molecular dynamics simulations. Glycine- and serine-rich amino acid fragments, common in flexible protein regions, play essential roles in functional properties. Results show excellent agreement between end-to-end distances, orientational factors from simulations, and the available experimental and theoretical data, validating the reliability of the GROMOS force field model. The end-to-end distribution, modeled using three Gaussian distributions, reveals a complex shape, confirmed by cluster analysis highlighting a limited number of significant conformations dominating the peptide landscape. All peptides predominantly adopt a disordered state in the solvent, yet exhibit a compact shape, aligning with the model of disordered polypeptide chains in poor solvents. Conformations show marginal dependence on chain composition, with Ser-only chains exhibiting slightly more elongation. This study enhances our understanding of peptide behavior, providing valuable insights into their structural dynamics in solution.

Measuring sub-nanometer undulations at microsecond temporal resolution with metal- and graphene-induced energy transfer spectroscopy

Out-of-plane fluctuations, also known as stochastic displacements, of biological membranes play a crucial role in regulating many essential life processes within cells and organelles. Despite the availability of various methods for quantifying membrane dynamics, accurately quantifying complex membrane systems with rapid and tiny fluctuations, such as mitochondria, remains a challenge. In this work, we present a methodology that combines metal/graphene-induced energy transfer (MIET/GIET) with fluorescence correlation spectroscopy (FCS) to quantify out-of-plane fluctuations of membranes with simultaneous spatiotemporal resolution of approximately one nanometer and one microsecond. To validate the technique and spatiotemporal resolution, we measure bending undulations of model membranes. Furthermore, we demonstrate the versatility and applicability of MIET/GIET-FCS for studying diverse membrane systems, including the widely studied fluctuating membrane system of human red blood cells, as well as two unexplored membrane systems with tiny fluctuations, a pore-spanning membrane, and mitochondrial inner/outer membranes.

Monitoring Real-Time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism.

RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.

An accurate probabilistic step finder for time-series analysis

Noisy time-series data—from various experiments, including Förster resonance energy transfer, patch clamp, and force spectroscopy, among others—are commonly analyzed with either hidden Markov models or step-finding algorithms, both of which detect discrete transitions. Hidden Markov models, including their extensions to infinite state spaces, inherently assume exponential—or technically geometric—holding time distributions, biasing step locations toward steps with geometric holding times, especially in sparse and/or noisy data. In contrast, existing step-finding algorithms, while free of this restraint, often rely on ad hoc metrics to penalize steps recovered in time traces (by using various information criteria) and otherwise rely on approximate greedy algorithms to identify putative global optima. Here, instead, we devise a robust and general probabilistic (Bayesian) step-finding tool that neither relies on ad hoc metrics to penalize step numbers nor assumes geometric holding times in each state. As the number of steps themselves in a time-series are a priori unknown, we treat these within a Bayesian nonparametric (BNP) paradigm. We find that the method developed, BNP Step (BNP-Step), accurately determines the number and location of transitions between discrete states without any assumed kinetic model and learns the emission distribution characteristic of each state. In doing so, we verify that BNP-Step can analyze sparser data sets containing higher noise and more closely spaced states than otherwise resolved by current state-of-the-art methods. What is more, BNP-Step rigorously propagates measurement uncertainty into uncertainty over state transition locations, numbers, and emission levels as characterized by the posterior. We demonstrate the performance of BNP-Step on both synthetic data as well as data drawn from force spectroscopy experiments.

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.

First-principles simulation of excitation energy transfer and transient absorption spectroscopy in the CP29 light-harvesting complex.

The dynamics of delocalized excitons in light-harvesting complexes (LHCs) can be investigated using different experimental techniques, and transient absorption (TA) spectroscopy is one of the most valuable methods for this purpose. A careful interpretation of TA spectra is essential for the clarification of excitation energy transfer (EET) processes occurring during light-harvesting. However, even in the simplest LHCs, a physical model is needed to interpret transient spectra as the number of EET processes occurring at the same time is very large to be disentangled from measurements alone. Physical EET models are commonly built by fittings of the microscopic exciton Hamiltonians and exciton-vibrational parameters, an approach that can lead to biases. Here, we present a first-principles strategy to simulate EET and transient absorption spectra in LHCs, combining molecular dynamics and accurate multiscale quantum chemical calculations to obtain an independent estimate of the excitonic structure of the complex. The microscopic parameters thus obtained are then used in EET simulations to obtain the population dynamics and the related spectroscopic signature. We apply this approach to the CP29 minor antenna complex of plants for which we follow the EET dynamics and transient spectra after excitation in the chlorophyll b region. Our calculations reproduce all the main features observed in the transient absorption spectra and provide independent insight on the excited-state dynamics of CP29. The approach presented here lays the groundwork for the accurate simulation of EET and unbiased interpretation of transient spectra in multichromophoric systems.

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.

Direct Comparison between Förster Resonance Energy Transfer and Light-Induced Triplet-Triplet Electron Resonance Spectroscopy.

To carry out reliable and comprehensive structural investigations, the exploitation of different complementary techniques is required. Here, we report that dual triplet-spin/fluorescent labels enable the first parallel distance measurements by electron spin resonance (ESR) and Förster resonance energy transfer (FRET) on exactly the same molecules with orthogonal chromophores, allowing for direct comparison. An improved light-induced triplet-triplet electron resonance method with 2-color excitation is used, improving the signal-to-noise ratio of the data and yielding a distance distribution that provides greater insight than the single distance resulting from FRET.

A system of FRET dyes designed to assess the degree of nano-scale contact between surfaces for interfacial adhesion

HypothesisInterfacial adhesion caused by intermolecular forces only occur between surfaces at nano-scale contact (NSC), i.e., 0.1–0.4 nm and can be evaluated using Forster resonance energy transfer spectroscopy (FRET). For this, a suitable pair of fluorescent dyes must be selected, which spectroscopic properties will determine the FRET system performance. Here, we present a brand-new FRET dye system specifically designed to measure NSC in the distance range relevant for van-der-Waals and hydrogen bonding, i.e., below 1 nm. ExperimentsWe propose the FRET pair: 7-Amino-4-methyl-cumarin (C120) and 5(6)-Carboxy-2′,7′-dichlor-fluorescein (CDCF) with high quantum yield (QY, QYC120 = 0.91 and QYCDCF = 0.64) and a distance detection range of 0.6–2.2 nm (0.1 mM). Adhered-thin films with increasing NSC degrees are produced with ascending pressure from 1.5 to 150 bar. To validate the proposed FRET measurement, we are correlating the bonded films interfacial adhesion (separation energy) to the measured FRET intensity, indicating its degree of NSC. FindingsWe find that the proposed dyes are producing the desired FRET signal in adhered-thin films, for an interaction range of 0.6–2.2 nm, with high sensitivity due to the dye’s high quantum yields. The increasing adhesion in these films is only caused by its increase in NSC. We find that the adhesion strength, measured as the separation energy between the films, is correlated to the measured FRET signal. Hence, the introduced FRET system is accurately able to measure the degree of NSC between soft surfaces.

Metasurfaces-Driven Hyperspectral Imaging via Multiplexed Plasmonic Resonance Energy Transfer.

Obtaining single-molecular-level fingerprints of biomolecules and electron-transfer dynamic imaging in living cells are critically demanded in postgenomic life sciences and medicine. However, the possible solution called plasmonic resonance energy transfer (PRET) spectroscopy remains challenging due to the fixed scattering spectrum of a plasmonic nanoparticle and limited multiplexing. Here, multiplexed metasurfaces-driven PRET hyperspectral imaging, to probe biological light-matter interactions, is reported. Pixelated metasurfaces with engineered scattering spectra are first designed over the entire visible range by the precision nanoengineering of gap plasmon and grating effects of metasurface clusters. Pixelated metasurfaces are created and their full dark-field coloration is optically characterized with visible color palettes and high-resolution color printings of the art pieces. Furthermore, three different biomolecules (i.e., chlorophyll a, chlorophyll b, and cytochrome c) are applied on metasurfaces for color palettes to obtain selective molecular fingerprint imaging due to the unique biological light-matter interactions with application-specific biomedical metasurfaces. This metasurface-driven PRET hyperspectral imaging will open up a new path for multiplexed real-time molecular sensing and imaging methods.

Combined experimental/computational aspects for the molecular interaction of empagliflozin with bovine serum albumin: Quantification and application to human plasma.

Empagliflozin (EMP) is an oral antihyperglycemic agent for type 2 diabetic patients. The molecular binding of EMP to bovine serum albumin (BSA) was elucidated by a combined experimental/computational approach to fulfil the pharmacokinetics and pharmacodynamics gaps of the cited drug for further development. Fluorescence, synchronous, and three-dimensional fluorescence spectroscopy verified that EMP quenched BSA native fluorescence through a dual static/dynamic mechanism that was further supported by Fӧrster resonance energy transfer and ultraviolet absorption spectroscopy. Fourier transform infrared spectroscopy revealed the conformational variations in BSA secondary structure induced by EMP. Thermodynamic properties of the BSA-EMP complex were also investigated, and the hydrophobic interactions' role in the binding process was demonstrated by the computed enthalpy (ΔH =6.558 kJ mol-1 ) and entropy (ΔS = 69.333 J mol-1 K-1 ). Gibbs free energy (ΔG) values were negative at three distinct temperatures, illuminating the spontaneity of this interaction. In addition, molecular docking studies depicted the optimal fitting of EMP to BSA on Site I (sub-domain IIA) through three hydrogen bonds. Additionally, and based on the quenching effect of EMP on BSA fluorescence, this study suggests a simple validated spectrofluorometric method for the quantitation of the studied drug in bulk form and human plasma samples with reasonable recoveries (96.99-103.10%).

Intracellular Conformation of Amyotrophic Lateral Sclerosis-Causative TDP-43.

Transactive response element DNA/RNA-binding protein 43 kDa (TDP-43) is the causative protein of amyotrophic lateral sclerosis (ALS); several ALS-associated mutants of TDP-43 have been identified. TDP-43 has several domains: an N-terminal domain, two RNA/DNA-recognition motifs, and a C-terminal intrinsically disordered region (IDR). Its structures have been partially determined, but the whole structure remains elusive. In this study, we investigate the possible end-to-end distance between the N- and C-termini of TDP-43, its alterations due to ALS-associated mutations in the IDR, and its apparent molecular shape in live cells using Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). Furthermore, the interaction between ALS-associated TDP-43 and heteronuclear ribonucleoprotein A1 (hnRNP A1) is slightly stronger than that of wild-type TDP-43. Our findings provide insights into the structure of wild-type and ALS-associated mutants of TDP-43 in a cell.

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.