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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.

Interfacial charge transfer dynamics in dye-sensitized NiO nanoparticles are being investigated for photocathodes in p-type dye-sensitized solar cells. In the photoreaction, after fast electron transfer from NiO to a molecule, the recombination of the hole in the nanoparticles with the electron in a reduced molecule plays an important role in the charge separation process and solar energy harvesting. Nevertheless, knowledge of the interfacial charge recombination (CR) rate and its mechanism is still limited due to the complex photoinduced electron and hole dynamics and lack of characterization of the inhomogeneity of the dynamics. Here, we report our work on probing interfacial charge recombination dynamics in Zn(ii)-5,10,15,20-tetra(3-carboxyphenyl)porphyrin (m-ZnTCPP) dye-sensitized NiO nanoparticles by correlating single-molecule fluorescence blinking dynamics with charge transfer dynamics using single-molecule photon-stamping spectroscopy. The correlated analyses of single-molecule fluorescence intensity, lifetime, and blinking reveal the intrinsic distribution and temporal fluctuation of interfacial charge transfer reactivity, which are closely related to site-specific molecular interactions and dynamics.

Factors controlling the interfacial electron-transfer (ET) dynamics in molecule–semiconductor systems have been intensively investigated in recent years. Here, we study dynamics of interfacial ET on Zn(II) meso-tetra (N-methyl-4-pyridyl) porphine tetrachloride (ZnTMPyP)–TiO2 nanoparticle (NP) system using single-molecule photon-stamping spectroscopy while electrostatically controlling the coupling between ZnTMPyP and TiO2 NP by changing the surface charge of the TiO2 NP. The single-molecule fluorescence trajectories show strong fluctuation and blinking between bright and dark states providing clear indication for the binding affinity between ZnTMPyP and the TiO2 NP via electrostatic interaction. By changing the surface charge on the TiO2 NP, positive or negative, we are able to change the coupling between ZnTMPyP and the TiO2 NP, which is revealed from the dominant dark states distribution in fluorescence trajectories and shorter fluorescence lifetime of ZnTMPyP attached on negatively charged TiO2 NP surf...

We investigate the surface-enhanced Raman scattering (SERS) spectra of flavin monoucleotide (FMN) in its different redox states and in a redox active enzyme, nitric oxide synthase. Incubated with silver nanoparticles coated with silica, spectra for oxidized and reduced FMN are obtained at different electrochemical potentials. Dominate Raman mode shifts at 1623/1610, 1567/1550, and 1502/1492 cm−1, belonging to typical redox-sensitive region of FMN, are observed and analyzed, and they show a consistence with the results of spectral calculation by using the density function theory (DFT) method. We assign mode at 1500 cm−1, composed of N5―H bending, N1C10a stretching and the asymmetric C4a―N5―C5a stretching, as a spectroscopic indicator for the redox states of FMN, because it shows a significant downshift (1502/1492 cm−1) and non-linearly correction with potentials when FMN gets reduced from its full oxidized state electrochemically. Isolated FMN domain from a wild type neuronal nitric oxide synthase enzyme (nNOS) is also studied with the same experimental approach. We have observed similar Raman mode down-shifting as that of the pure FMN, which further supports our conclusion that the mode at ~1500 cm−1 indicates redox states for FMN, the coenzyme of nNOS, even in redox protein matrix. Copyright © 2016 John Wiley & Sons, Ltd.

Photoinduced, interfacial electron transfer (ET) dynamics between m-ZnTCPP and Sn-doped In2O3 (ITO) film has been studied using single-molecule photon-stamping spectroscopy. The observed ET dynamics of single m-ZnTCPP adsorbed on ITO was compared with that of m-ZnTCPP adsorbed on TiO2 NPs with and without applied electric potential. Compared to m-ZnTCPP on the TiO2 NP surface, m-ZnTCPP on the ITO surface shows a reduced lifetime as well as suppressed blinking and a quasi-continuous distribution of fluorescence intensities, presumably due to higher electron density in ITO. The higher electron density leads to the occupancy of CB acceptor states/trap states, which supports a higher backward electron transfer (BET) rate that results in a quasi-continuous distribution of fluorescence intensities. The dependence of BET rate on electron density and charge trapping is consistent with our previous observations of quasi-continuous distribution of fluorescence intensities of m-ZnTCPP on TiO2 NPs with applied negative potential across the dye-TiO2 interface. The quasi-continuous distribution of fluorescence intensities in both cases of m-ZnTCPP on the ITO surface and m-ZnTCPP on TiO2 NPs with applied negative potential indicates that the electron density in the semiconductor plays a dominant role in dictating the changes in rates of charge transfer, rather than the relative energetics between electrons in the semiconductor and the oxidized sensitizer.

Tracking the energy flow in nanoscale materials is an important yet challenging goal. Experimental methods for probing the intermolecular energy transfer (ET) are often burdened by the spectral crosstalk between donor and acceptor species, which complicates unraveling their individual contributions. This issue is particularly prominent in inorganic nanoparticles and biological macromolecules featuring broad absorbing profiles. Here, we demonstrate a general spectroscopic strategy for measuring the ET efficiency between nanostructured or molecular dyes exhibiting a significant donor-acceptor spectral overlap. The reported approach is enabled through spectral shaping of the broadband excitation light with solutions of donor molecules, which inhibits the excitation of respective donor species in the sample. The resulting changes in the acceptor emission induced by the spectral modulation of the excitation beam are then used to determine the quantum efficiency and the rate of ET processes between arbitrary fluorophores (molecules, nanoparticles, polymers) with high accuracy. The feasibility of the reported method was demonstrated using a control donor-acceptor system utilizing a protein-bridged Cy3-Cy5 dye pair and subsequently applied for studying the energy flow in a CdSe

Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental understanding of the enzymatic reaction turnover dynamics associated with overall enzyme as well as the specific active-site conformational fluctuations that are not identifiable and resolvable in the conventional ensemble-averaged experiment.

The photosensitized interfacial electron transfer (ET) dynamics of the zinc(II)–5,10,15,20-tetra(3-carboxyphenyl)porphyrin (m-ZnTCPP)–TiO2 nanoparticle (NP) system has been studied using single-molecule photon-stamping spectroscopy. The single-molecule fluorescence intensity trajectories of m-ZnTCPP on TiO2 NP surface show fluctuations and blinking between bright and dark states, which are attributed to the variations in the reactivity of interfacial ET, i.e., intermittent interfacial electron transfer dynamics. Comparing the results with that from our earlier studied p-ZnTCPP–TiO2 nanoparticle system, we show the effect of anchoring group binding geometry (meta or para), hence electronic coupling of sensitizer (m-/p-ZnTCPP) and TiO2 substrate, on interfacial ET dynamics. Compared to p-ZnTCPP on TiO2 NP surface, with m-ZnTCPP, dark states are observed to dominate in single-molecule fluorescence intensity trajectories. This observation coupled with the large difference in lifetime derived from bright and d...

The misfolding and aggregation of amyloid β (1-42) peptide is crucial for a mechanical understanding of the formation of Alzheimer's disease. To investigate the detailed aggregation pathway and mechanism, it is important to identify the secondary structures of different aggregation forms. Here, we report probing different amyloid β aggregations in real time by using correlated approaches such as shell-isolated surface-enhanced Raman spectroscopy, thioflavin T fluorescence assay, and atomic force microscopy imaging. Our experimental results of Raman shifts have been further demonstrated by theoretical calculation, which indicates that the Raman spectral fingerprint changes are originated from the amyloid β secondary structure changes. Copyright © 2013 John Wiley & Sons, Ltd.

We investigate single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic and highly soluble Cr(VI) can be efficiently reduced to the less toxic and non-soluble Cr2O3 nanoparticles by MR-1. Cr2O3 is observed to emerge as nanoparticles adsorbed on the cell surface and its chemical nature is identified by EDX imaging and Raman spectroscopy. Co-localization of Cr2O3 and cytochromes by EDX imaging and Raman spectroscopy suggests a terminal reductase role for MR-1 surface-exposed cytochromes MtrC and OmcA. Our experiments revealed that the cooperation of surface proteins OmcA and MtrC makes the reduction reaction most efficient, and the sequence of the reducing reactivity of the MR-1 is: wild type > single mutant ΔmtrC or mutant ΔomcA > double mutant (ΔomcA-ΔmtrC). Moreover, our results also suggest that the direct microbial Cr(VI) reduction and Fe(II) (hematite)-mediated Cr(VI) reduction mechanisms may co-exist in the reduction processes.

Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area, and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3–Cy5 labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling.

The inhomogeneous interfacial electron transfer (IET) dynamics of 9-phenyl-2,3,7-trihydroxy-6-fluorone (PF)-sensitized TiO(2) nanoparticles (NPs) has been probed by a single-molecule photon-stamping technique as well as ensemble-averaged femtosecond transient absorption spectroscopy. The forward electron transfer (FET) time shows a broad distribution at the single-molecule level, indicating the inhomogeneous interactions and ET reactivity of the PF/TiO(2) NP system. The broad distribution of the FET time is measured to be 0.4 +/- 0.1 ps in the transient absorption and picoseconds to nanoseconds in the photon-stamping measurements. The charge recombination time, having a broad distribution at the single-molecule level, clearly shows a biexponential dynamic behavior in the transient absorption: a fast component of 3.0 +/- 0.1 ps and a slow component of 11.5 +/- 0.5 ns. We suggest that both strong and weak interactions between PF and TiO(2) coexist, and we have proposed two mechanisms to interpret the observed IET dynamics. A single-molecule photon-stamping technique and ensemble-averaged transient absorption spectroscopy provide efficient "zoom-in" and "zoom-out" approaches for probing the IET dynamics. The physical nature of the observed multiexponential or stretched-exponential ET dynamics in the ensemble-averaged experiments, often associated with dynamic and static inhomogeneous ET dynamics, can be identified and analyzed by single-molecule spectroscopy measurements.

How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting protein, LH2, can exist as linear aggregates of 4 +/- 2 proteins in the photosynthetic membranes and that the protein distributions are highly heterogeneous. In the photosynthetic membranes examined in our measurements, the ratio of the aggregated to the nonaggregated LH2 proteins is about 3:1 to 5:1 depending on the intensity of the illumination used during sample incubation and on the bacterial species. AFM images further identify that the LH2 proteins in the linear aggregates are monotonically tilted at an angle 4 +/- 2 degrees from the plane of the photosynthetic membranes. The aggregates result in red-shifted absorption and emission spectra that are measured using various mutant membranes, including an LH2 knockout, LH1 knockout, and LH2 at different population densities. Measuring the fluorescence lifetimes of purified LH2 and LH2 in membranes, we have observed that the LH2 proteins in membranes exhibit biexponential lifetime decays whereas the purified LH2 proteins gave single exponential lifetime decays. We attribute that the two lifetime components originate from the existence of both aggregated and nonaggregated LH2 proteins in the photosynthetic membranes.

Site-specific spectroscopy is critical for a molecular-level understanding of the mechanisms and dynamics of the inhomogeneous chemical processes crucial for catalysis, surface and interfacial chemistry, and membrane protein dynamics in living cells. Surface-enhanced Raman scattering (SERS) spectroscopy and microscopy combined with atomic force microscopy (AFM) using metal-coated AFM tips have proven to be powerful in spectroscopic analysis of inhomogeneous processes, providing correlated topographic and spectroscopic information at the nanoscale from sites in highly heterogeneous environments. It has recently been observed that SERS spectral fluctuations are pertinent to site-specific spectroscopy and microscopy. Such fluctuations are important in that they hold promise for the study of molecular structure and dynamics at a single-molecule level. This article reviews our recent work on characterization and analysis of SERS spectral fluctuation dynamics at the nanoscale in metallic interstitial sites. Fluctuations were found to accompany nanoscale confined electromagnetic near-field enhancement. The result of such confinement is that only a few molecules dominate the far-field SERS spectral signal detected in microscopic measurements that probe one nanoscale 'hot' site at a time. The fluctuation amplitude significantly decreased with the number of molecules confined at the nanoscale-local field. A new AFM-coupled two-channel photon time-stamping system, enabling in situ correlation of the topographic and spectroscopic information for single nanoparticle clusters, was used to record the Raman intensity fluctuation trajectories at a sub-microsecond resolution. Experimentally, we found that SERS fluctuation dynamics are highly inhomogeneous amongst nanocluster interstitial sites although molecular translational and rotational motions at the interstitial sites can account for the SERS spectral fluctuations. To further understand these fluctuations at the nanoscale interstitial sites and nanostructures, field enhancement and field distribution at different interstitial site topographies and rough fractal surfaces were studied using finite element method computational simulation in a classic electrodynamics approach.

We demonstrate the use of single-molecule spectroscopy to study enzyme conformational motions of T4 lysozyme under hydrolysis reaction of the polysaccharide walls of E. coli B cells. By attaching a donor−acceptor pair of dye molecules site-specifically to noninterfering sites on the enzyme, the hinge-bending motions of the enzyme are measured by monitoring the donor−acceptor emission intensity as a function of time. The overall enzymatic reaction rate constants are found to vary widely from molecule to molecule. The dominant contribution to this static inhomogeneity is attributed to enzyme searching for reactive sites on the substrate. We have also applied molecular dynamics simulation and a random-walk model to analyze the enzyme−substrate complex formation dynamics, revealing multiple intermediate conformational states in the chemical reaction process. This approach provides information about the microscopic conformational change drifting velocity, diffusion coefficient, friction coefficient, energy con...

Soluble copper-based M4L4 lantern-type metal–organic cages bearing internal amines were synthesized. The solution state integrity of the paramagnetic metal–organic cages was demonstrated using NMR, DLS, MS, and AFM spectroscopy. 1D supramolecular pillars of pre-formed cages or covalent host–guest complexes selectively formed upon treatment with 4,4′-bipyridine and acetic anhydride, respectively.

An approach to probing single-molecule ion channel kinetics and conformational dynamics, patch-clamp confocal fluorescence microscopy (PCCFM), uses simultaneous ultrafast fluorescence spectroscopy and single-channel electric current recording. PCCFM is applied to determine single-channel conformational dynamics by probing single-pair fluorescence resonant energy transfer, fluorescence self-quenching, and anisotropy of the dye-labeled gramicidin ion channel incorporated in an artificial lipid bilayer. Hidden conformational changes were observed, which strongly suggests that multiple intermediate conformation states are involved in gramicidin ion channel dynamics.

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

In 1959, Nobel laureate Richard Feynman suggested that ‘there is plenty of room at the bottom,’ predicting the possibility of single-molecule detection and studies. Over the last 20 years, we have witnessed a rapid development in single-molecule spectroscopy. Singlemolecule spectroscopy and imaging have been demonstrated to be a powerful molecular analytical approach to studying the complex and inhomogeneous chemical, biological, and physical processes involved in protein dynamics, protein-protein interactions, protein-DNA interaction dynamics, biological and chemical catalyses, and interfacial dynamics. The discipline of singlemolecule spectroscopy has been expanded across a broad range, including optical imaging, optical spectroscopy of fluorescence and Raman, atomic force spectroscopy, and various forms of scanning probe microscopy. The significant character of this exciting development is that single-molecule spectroscopy is developing hand-in-hand with the recent advancements in nanotechnology, imaging technologies, ultrafast dynamics technologies, theoretical modeling and analyses, and computational technologies. (Published: 22 February 2010) Citation: Nano Reviews 2010, 1 : 5052 - DOI: 10.3402/nano.v1i0.5052

Enzymatic reactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. Protein-molecular interactions define the enzymatic reaction potential surface, pathway, and dynamics. The single-molecule protein-protein interaction dynamics reveals the nature of the molecular complex formation and recognition that are critical for an enzymatic reaction to occur. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. We obtained the rates for single-molecule conformational active-site open-close fluctuation and correlated enzymatic reactions. We have demonstrated a specific statistical analysis to reveal single-molecule FRET anti-correlated fluctuations from a high background of fluorescence correlated thermal fluctuations. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels. Such complexity makes it difficult, if not impossible, to characterize ion channel dynamics using conventional electrical recording alone since that the measurement does not specifically interrogate the associated conformational changes but rather the consequences of the conformational changes. Recently, new technology developments on single-molecule spectroscopy, and especially, the combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging have provided us the capability of probing ion channel conformational changes simultaneously with the electrical single channel recording. The function-regulating and site-specific conformational changes of ion channels are now measurable under physiological conditions in real-time, one molecule at a time. In this chapter, we will focus our discussion on the new development of real-time imaging of the dynamics of individual ion channels using a novel combination of single-molecule fluorescence spectroscopy and single-channel current recordings. We will then discuss a specific example of single-molecule gramicidin ion channel dynamics studied by the new approach and the future prospects.

Redox protein nanoscale domains on the cell surface of a bacterium, Shewanella oneidensis MR1, grown in the absence and presence of electron acceptors, is topographically characterized using combined atomic force microscopy (AFM) and confocal surface enhanced Raman scattering (SERS) spectroscopy. The protruding nanoscale domains on the outer membrane of S. oneidensis were observed, as was their disappearance upon exposure to electron acceptors such as oxygen, nitrate, fumarate, and iron nitrilotriacetate (FeNTA). Using SERS spectroscopy, a redox heme protein was identified as a major component of the cell surface domains. This conclusion was further confirmed by the disappearance of Raman vibrational frequencies, characteristic of heme proteins, upon exposure of the cells to electron acceptors. Our experimental results from our AFM imaging and SERS spectroscopy, consistent with the literature, suggest the protruding nanoscale surface domains as heme-containing secretions. Our results on the distributions of redox proteins on microbial cell surfaces will be helpful for a mechanistic understanding of the behaviors of surface proteins and their interactions with redox environments.

We have probed single-molecule metal-to-ligand charge transfer (MLCT) dynamics of a ruthenium complex at room temperature. Using photon antibunching measurements under continuous wave (CW) laser excitation, nonclassical photon statistics, and excitation power dependent measurements, we were able to selectively measure the single-molecule MLCT state lifetime. This work demonstrated, as the first single-molecule photon antibunching measurement of the triplet excited state, a new application of single-molecule spectroscopy on excited-state dynamics and ground-state recovering dynamics of an important class of chemical species that have often been used and studied in energy conversion and electron transfer.

Protein conformational fluctuations and dynamics, often complex and associated with inhomogeneities, play a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the proteins are involved in multiple-step, multiple-conformation complex chemical interactions and transformations, such as in enzymatic reactions, protein-protein interactions, and ion-channel membrane protein processes. Alternatively, single-molecule spectroscopy is a powerful approach to probing and analyzing protein conformational dynamics in real time.

Protein-protein and protein-DNA interactions play critical roles in biological systems, and these interactions often involve complex mechanisms and inhomogeneous dynamics. Single-molecule spectroscopy is a powerful and complimentary approach to decipher such spatially and temporally inhomogeneous protein interaction systems, providing new information that are not obtainable from static structure analyses, thermodynamics characterization, and ensemble-averaged measurements. To illustrate the single-molecule spectroscopy and imaging technology and their applications on studying protein-ligand interactions, this chapter focuses on discussing two recent single-molecule spectroscopy studies on protein-protein interaction in cell signaling process and on protein-DNA interactions in DNA damage recognition process.

Protein conformational dynamics, often associated with static and dynamic inhomogeneities, plays a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the protein involves in a multiple-step complex chemical reaction, such as an enzymatic reaction. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics in real time under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. In this minireview, we discuss our recent studies on single-molecule enzymatic reaction dynamics and protein conformational dynamics of the T4 lysozyme hydrolyzation reaction of a polysaccharide by a combined approach of single-molecule spectroscopy, molecular dynamics simulation, and theoretical modeling. Detailed characterization of the complex enzymatic reaction dynamics identified the nature of the inhomogeneity and revealed multiple intermediate conformational states associated with the enzyme-substrate complex formation in the multiple-step enzymatic reaction.

How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting proteins, LH2, can exist as linear aggregates of 2 to 8 proteins in the photosynthetic membranes, and the protein distributions are highly heterogeneous. LH2 antenna proteins are responsible for absorbing most of the light energy for photosynthesis, and efficient intra- and inter-molecular energy transfers of LH2 complexes are important for the overall efficiency of the light harvesting mechanism. We combined AFM imaging and spectroscopic analysis with J aggregate theoretical calculations to characterize the linear aggregation of LH2. AFM images reveal the linear aggregation of LH2, where the LH2 complexes are tilted to the plane of the photosynthetic membrane. The spectroscopic results support the attribution of LH2 complexes in the membrane to linear aggregates. The calculated values for the absorption, emission and lifetime using a model developed from J aggregate theory are consistent with the experimentally determined spectroscopic values, further proving a J type aggregation of the LH2 complexes in the photosynthetic membrane.

Single-molecule conformational manipulation provides unique methods for studying the relationship between function and structure of biomolecules, and for exploring novel properties of biomolecules under complex local environments. Enzymatic reactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. We have developed and applied AFM/Magnetic Tweezers force manipulation combined single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules under pico-Newton force manipulations were observed simultaneously in real-time by single-molecule FRET detections. By a repetitive pulling-releasing manipulation of a Cy3-Cy5 dye labeled kinase molecules under the conditions with and without enzymatic substrates, we observed and analyzed the enzymatic conformational dynamics. We demonstrated that the enzyme conformational flexibility can be regulated by enzyme-substrate interaction, an experimental evidence of protein-substrate folding-binding interaction mechanism. Our new approach is applicable to a wide range of single-molecule AFM-FRET measurements for protein conformational changes under enzymatic reactions, including controls of enzymatic reactivity by mechanical-force manipulating protein conformations.Reference:1. Qing Guo, Yufan He, H. Peter Lu, “Repetitive Force Pulling-Releasing Manipulation of Enzymatic Conformational Fluctuations and Enzyme-Substrate Interactions by Single-Molecule FRET-Magnetic Tweezers Microscopy,” Phys. Chem. Chem. Phys., 16, 13052 −13058 (2014).2. H. Peter Lu, “Sizing up single-molecule enzymatic conformational dynamics,” Chem. Soc. Rev., Invited Review Article, 43, 1118-1143 (2014).3. Yufan He, Maolin Lu, H. Peter Lu, “Single-Molecule Photon Stamping FRET Spectroscopy Study of Enzymatic Conformational Dynamics,” Phys. Chem. Chem. Phys., 15, 770-775 (2013) (Cover page).4. Yufan He, Maolin Lu, Jin Cao, H. Peter Lu, “Manipulating Protein Conformations by Single-Molecule AFM-FRET Nanoscopy,” ACS Nano, 6, 1221-1229 (2012).

We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscopy measurements. This integrated single-molecule spectroscopy is powerful in studies of single molecule dynamics at interfaces of biological and chemical systems.

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.

How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. Their specific architecture helps them to absorb photons in the primary process of photosynthesis that is subsequently followed by a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. In this work, using atomic force microscopy (AFM) imaging, single membrane fragment FRET spectroscopy, spectral fluctuation analysis, and time-resolved spectroscopic analysis, we show the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions. Our results suggest that the light harvesting proteins, LH2, mostly exist in two states, the aggregated and non-aggregated states in the photosynthetic membranes. Our results shed a light on understanding the complex intramolecular energy transfer dynamics and mechanism of the light harvesting in the photosynthetic membranes.