Single-Molecule Human Nucleosome Spontaneously Ruptures under the Stress of Compressive Force: A New Perspective on Gene Stability and Epigenetic Pathways.

Force manipulation on the biological entities from living cells to protein molecules has revealed many mechanical details of cell biology from resolving folding and unfolding pathways to finding molecular interaction forces. A nucleosome is the basic repeating unit of chromatin where the histone octamer is wrapped by DNA, important for gene stability and regulation. How the inner side of the DNA gets accessed by other DNA binding molecules has been a puzzle that has been intensively studied and debated, important to epigenetics, gene stability, and regulations. Here we report our observation of spontaneous ruptures of human nucleosomes under pico-Newton (pN) compressive force. The amplitude of the compressive force, a squeezing rather than pulling force, involved in our experiment is tens of pN, which can be thermally available by biological force fluctuation at room temperature and under physiological conditions. This kind of structural rupture can loosen up the DNA around the histone, which in turn makes the DNA accessible to transcription and epigenetic modifications.

A Missing Origin of the Tau Protein Aggregation Pathway Triggered by Thermal and Biological Forces.

Fluctuations in mechanical force vectors within living cells can substantially influence the behavior and functions of proteins. Tau protein can spontaneously be raptured and entangled in refolding under picoNewton compressive forces that are biologically available in a living cell: a hidden aggregation pathway due to stress and crowding. Our findings were achieved through a customized modification of atomic force microscopy (AFM) for single-molecule manipulation. This previously hidden phenomenon of proteins rupturing collectively while subsequently and spontaneously refolding into a complex entangled conformation, distinct from the Tau protein's folded or unfolded states, could potentially explain the early-event initiation of the aggregation of the Tau protein seen in various neurodegenerative diseases. This article introduces our recent discovery of the missing Tau protein property that is of significant relevance to the Tau protein and neurodegenerative disease research and medical treatment, aiming to stimulate the collective observation and a new perspective on the Tau aggregation mechanism and disease mechanism studies.

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.

Probing Protein-DNA Conformational Dynamics in DNA Damage Recognition: Xeroderma Pigmentosum Group A Stabilizes the Damaged DNA-RPA14 Complex by Controlling Conformational Fluctuation Dynamics.

DNA damage inside biological systems may result in diseases like cancer. One of the major repairing mechanisms is the nucleotide excision repair (NER) that recognizes and repairs the damage caused by several internal and external exposures, such as DNA double-strand distortion due to the chemical modifications. Recognition of lesions is the initial stage of the DNA damage repair, which occurs with the help of several proteins like Replication Protein A (RPA) and Xeroderma Pigmentosum group A (XPA). The recognition process involves complex conformational dynamics of the proteins. Studying the dynamics of damage recognition by these proteins helps us to understand the mechanism and to develop therapeutics to increase the efficiency of recognition. Here, we use single-molecule fluorescence fluctuation measurements of a dye, labeled at a damaged position on DNA, to understand the interaction of the damage site with RPA14 and XPA. Our results suggest that interactive conformational dynamics of RPA14 with damaged DNA is inhomogeneous due to its low affinity for DNA, whereas binding of XPA with the already formed DNA-RPA14 complex may increase the specificity of damage recognition by controlling the conformational fluctuation dynamics of the complex.

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.

Compressive-force induced activation of apo-calmodulin in protein signalling.

Mechanical force plays a critical role in the relationship between protein structure and function. Force manipulation by Atomic Force Microscope can be significant and trigger chemical and biological activities of proteins. Previously we have reported that Apo-CaM undergoes through a spontaneous tertiary structural rupture under a piconewton compressive force. Here we have observed that the ruptured Apo-CaM molecules can be available to bind with C28W peptide, a typical protein signalling activity that only a Ca2+-activated CaM has. This behaviour is both unexpected and profound, as CaM in its Ca2+-non-activated form has a closed structure which does not presumably allow the molecule to bind to target peptides. In this experiment, we demonstrate that both chemical activation and force activation can play a vital role in biology, such as the cell-signalling protein dynamics and function.

Spontaneous Rupture and Entanglement of Human Neuronal Tau Protein Induced by Piconewton Compressive Force.

Mechanical force vector fluctuations in living cells can have a significant impact on protein behavior and functions. Here we report that a human tau protein tertiary structure can abruptly and spontaneously rupture, like a balloon, under biologically available piconewton compressive force, using a home-modified atomic force microscopy single-molecule manipulation. The rupture behavior is dependent on the physiological level of presence of ions, such as K+ and Mg2+. We observed rupture events in the presence of K+ but not in the presence of Mg2+ ions. We have also explored the entangled protein state formed following the events of the multiple and simultaneous protein ruptures under crowding. Crowded proteins simultaneously rupture and then spontaneously refold to an entangled folding state, different from either folded and unfolded states of the tau protein, which can be a plausible pathway for the tau protein aggregation that is related to a number of neurodegenerative diseases.

Raman Spectroscopic Analysis of Signaling Molecules-Dopamine Receptors Interactions in Living Cells.

The selective interaction of signaling compounds including neurotransmitters and drugs with the dopamine receptors (DARs) is extremely important for the treatment of neurodegenerative diseases. Here, we report a method to probe the selective interactions of signaling compounds with D1 and D2 DARs in living cells using the combined approach of theoretical calculation and surface-enhanced Raman spectroscopy (SERS). When signaling compounds such as DA, amphetamine, methamphetamine, and methylenedioxypyrovalerone interact with D1 dopamine receptors (DRD1), the intracellular cyclic adenosine monophosphate (cAMP) level is increased. However, the intracellular level of cAMP is decreased when D2 dopamine receptors (DRD2) interact with the abovementioned signaling compounds. In our experiments, we have internalized the silica-coated silver nanoparticles (AgNP@SiO2) in living cells to adsorb biologically generated cAMP which was probed by using SERS. Besides adsorptions of cAMP, AgNP@SiO2 has a crucial role for the enhancement of Raman cross section of the samples. We observed the characteristic SERS peaks of cAMP when DRD1-overexpressed cells interact with the signaling compounds; these peaks were not observed for other cells including DRD2-overexpressed and DRD1-DRD2-coexpressed cells. Our experimental approach is successful to probe the intracellular cAMP and characterize the selectivity of signaling compounds to different types of DARs. Furthermore, our experimental approach is highly capable for in vivo studies because it can probe intracellular cAMP using a low input power of incident laser without significant cell damage. Our experimental results and density functional theory calculations showed that 780 and 1503 cm-1 are signature Raman peaks of cAMP. The SERS peak at 780 cm-1 is associated with C-O, C-C, and C-N stretching and symmetric and asymmetric bending of two O-H bonds of cAMP, whereas the SERS peak at 1503 cm-1 is contributed by the O9-H3 bending mode.

Oscillating Piconewton Force Manipulation on Single-Molecule Enzymatic Conformational and Reaction Dynamics.

Oscillation force has been demonstrated in theoretical studies as a critical role in unraveling the comprehensive enzymatic dynamics and addressing its regulation on enzyme activity. Utilizing the imposed external mechanical oscillation force by our newly developed magnetic tweezers coupled with a single-molecule photon-stamping imaging spectroscopic microscope, we experimentally studied a millisecond-scale oscillation force manipulation on single horseradish peroxidase enzymatic conformational and reaction dynamics. We have studied the enzymatic reaction dynamics and found that the enzyme activity changes under the real-time oscillatory force manipulation. Moreover, the oscillation force shows the capability of manipulating the enzyme active-site conformational state as well as the nascent-formed product's interaction with the active site of the enzyme, which impacts on the product release pathways. Specifically, we have identified that there are two product releasing pathways, the solvation-mediated diffusion releasing pathway and the spilling-out releasing pathway. We have observed that the spilling-out pathway can be significantly perturbed by the oscillatory force manipulation. Our correlated interpretation of enzymatic conformational and reaction dynamics provides a new insight into the comprehensive understanding of the complex conformational dynamics evolved in an enzymatic reaction. Technically, we have also demonstrated a novel approach capable of unfolding an enzyme under an enzymatic reaction condition in real time and, furthermore, by using an oscillatory mechanical weak piconewton force to manipulate enzyme conformations, and the enzyme thermal fluctuation is fully maintained. The real-time in situ fluorescence probe at the enzymatic active site reports the active-site conformational dynamics through each enzymatic reaction turnovers.

Mode-Selective Raman Imaging of Dopamine-Human Dopamine Transporter Interaction in Live Cells.

Dopamine (DA) is the catecholamine neurotransmitter which interacts with dopamine receptors (DARs) to generate dopaminergic signals in the nervous system. Dopamine transporter (DAT) interacts with DA to maintain DA's homeostasis in synaptic and perisynaptic space. DAT and DARs have great importance in the central nervous system (CNS) because they are associated with the targeted binding of drugs. Interactions of DA, its analogue with DARs, or DAT have been studied extensively to understand the mechanism of the dopaminergic signaling process and several neurodegenerative diseases, including schizophrenia, Parkinson's diseases, addiction, attention deficit hyperactivity disorder, and bipolar disorder. However, there is still a lack of a risk-free, label-free, and minimally invasive imaging approach to probe the interaction between DA and DAT or DARs. Here, we probed the DA, human dopamine transporter (hDAT), and DA-hDAT interactions in live cells using combined approach of two-photon excited (2PE) fluorescence imaging and mode-selective Raman measurement. We utilized the signature Raman peak at 1287 cm-1 to probe the location of DA and 807 and 1076 cm-1 to probe the DA-hDAT interaction in live cells. We found that the combined approach of mode-selective Raman imaging, 2PE fluorescence imaging, and computational methods is successful to probe and confirm the DA-hDAT interactions in living cells. The probing of the interactions of DARs or DAT with DA or other targeting drugs is crucial for the diagnosis and cure of several neurodegenerative diseases. Also, this analytical approach could be extended to probe other types of protein-ligand interactions.

Single-Molecule Spectroscopy Study of Crowding-Induced Protein Spontaneous Denature and Crowding-Perturbed Unfolding-Folding Conformational Fluctuation Dynamics.

The effects of molecular crowding on protein folding-unfolding processes are of importance for understanding protein function and structure dynamics in living cells. The enhancement of protein stability as a result of reduced entropic effect in the presence of molecular crowding is well understood both experimentally and theoretically. However, because of the complexity and interplay between various interactions existing in an equally favored environment of protein folding and unfolding conformational dynamics, such a simple reduced entropic enhancement model does not suffice to describe protein folding conformational dynamics under a protein crowding condition. In this paper, we report our observation on that single protein molecules spontaneously denature into unfolded proteins and folding-unfolding fluctuations in solution of crowding reagent Ficoll 70. We have identified that such protein dynamics involves a combined mechanism of polymer-polymer interaction, entropic effects, and protein solvation dynamics. We characterize the protein folding-unfolding dynamics by using single-molecule spectroscopy to obtain detailed molecular dynamic scale information on the protein folding-unfolding conformational fluctuation dynamics. Our findings suggest that the complex unfolding dynamic processes are spontaneous denature of single protein molecules induced by molecular crowding effect which has been elusive for analysis in ensemble-averaged measurements. Furthermore, the energy needed for the spontaneous unfolding is at the biological accessible force fluctuation level, which suggests a strong implication of significant human health relevance and importance. The new knowledge of the inhomogeneous protein unfolding processes can serve as a step forward to a mechanistic understanding of human diseases associated with molecular crowding, protein aggregates, fibril formation, as well as gene translational regulation processes typically under a molecular crowded local environment.

Ratiometric Near-Infrared Fluorescent Probes Based On Through-Bond Energy Transfer and π-Conjugation Modulation between Tetraphenylethene and Hemicyanine Moieties for Sensitive Detection of pH Changes in Live Cells.

In this paper, we present three ratiometric near-infrared fluorescent probes (A-C) for accurate, ratiometric detection of intracellular pH changes in live cells. Probe A consists of a tetraphenylethene (TPE) donor and near-infrared hemicyanine acceptor in a through-bond energy transfer (TBET) strategy, while probes B and C are composed of TPE and hemicyanine moieties through single and double sp2 carbon-carbon bond connections in a π-conjugation modulation strategy. The specific targeting of the probes to lysosomes in live cells was achieved by introducing morpholine residues to the hemicyanine moieties to form closed spirolactam ring structures. Probe A shows aggregation-induced emission (AIE) property at neutral or basic pH, while probes B and C lack AIE properties. At basic or neutral pH, the probes only show fluorescence of TPE moieties with closed spirolactam forms of hemicyanine moieties, and effectively avoid blind fluorescence imaging spots, an issue which typical intensity-based pH fluorescent probes encounter. Three probes show ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with TPE fluorescence decreases and hemicyanine fluorescence increases, because acidic pH makes the spirolactam rings open to enhance π-conjugation of hemicyanine moieties. However, probe A shows much more sensitive ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with remarkable ratio increase of TPE fluorescence to hemicyanine fluorescence up to 238-fold than probes B and C because of its high efficiency of energy transfer from TPE donor to the hemicyanine acceptor in the TBET strategy. The probe offers dual Stokes shifts with a large pseudo-Stokes shift of 361 nm and well-defined dual emissions, and allows for colocalization of the imaging readouts of visible and near-infrared fluorescence channels to achieve more precisely double-checked ratiometric fluorescence imaging. These platforms could be employed to develop a variety of novel ratiometric fluorescent probes for accurate detection of different analytes in applications of chemical and biological sensing, imaging, and diagnostics by introducing appropriate sensing ligands to hemicyanine moieties to form on-off spirolactam switches.

Probing Activated and Non-Activated Single Calmodulin Molecules under a Piconewton Compressive Force.

Interrogating the protein structure-function inter-relationship under a piconewton force manipulation has been highly promising and informative. Although protein conformational changes under pulling force manipulations have been extensively studied, protein conformational changes under a compressive force have not been explored in detail. Using our home-modified sensitive and high signal-to-noise atomic force microscopy (AFM) approach, we have applied a piconewton compressive force, manipulating a Calmodulin (CaM) molecule to characterize two different forms of CaM, the Ca2+-ligated activated form and the Ca2+ free non-activated form (apo-CaM). We observed sudden and spontaneous structural rupture of apo-CaM under compressive force applied by an AFM tip, though no such events were recorded in the case of Ca2+-ligated activated CaM form. The sudden spontaneous structural rupture under a piconewton force compression has never been reported before, which presents an unexplored function that is likely important for protein-protein interactions and cell signaling functions.

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach.

The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording. Identifying the compositions of NMDA receptors, their spatial organization and distributions over live cell membranes, we observe that NMDA receptors are organized inhomogeneously: nearly half of the receptor proteins are individually dispersed; whereas others exist in heterogeneous clusters of around 50 nm in size as well as co-localized within the diffraction limited imaging area. We demonstrate that inhomogeneous interactions and partitions of the NMDA receptors can be a cause of the heterogeneous gating mechanism of NMDA receptors in living cells. Furthermore, comparing the imaging results with the ion-channel electric current recording, we propose that the clustered NMDA receptors may be responsible for the variation in the current amplitude observed in the on-off two-state ion-channel electric signal trajectories. Our findings shed new light on the fundamental structure-function mechanism of NMDA receptors and present a conceptual advancement of the ion-channel mechanism in living cells.

Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under picoNewton Compressive Force Manipulation.

Manipulating protein conformations for exploring protein structure-function relationship has shown great promise. Although protein conformational changes under pulling force manipulation have been extensively studied, protein conformation changes under a compressive force have not been explored quantitatively. The latter is even more biologically significant and relevant in revealing protein functions in living cells associated with protein crowdedness, distribution fluctuations, and cell osmotic stress. Here we report our experimental observations on abrupt ruptures of protein native structures under compressive force, demonstrated and studied by single-molecule AFM-FRET spectroscopic nanoscopy. Our results show that the protein ruptures are abrupt and spontaneous events occurred when the compressive force reaches a threshold of 12-75 pN, a force amplitude accessible from thermal fluctuations in a living cell. The abrupt ruptures are sensitive to local environment, likely a general and important pathway of protein unfolding in living cells.

Exploration of Multistate Conformational Dynamics upon Ligand Binding of a Monomeric Enzyme Involved in Pyrophosphoryl Transfer.

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) is a monomeric protein with 158 residues, which undergoes large-scale conformational changes between apo, open, and holo states responding to ligand binding for its function. It has been explored widely as an excellent target for potential antibacterial drug development. However, little is known about how conformational dynamics between the native states influences the substrate recognition and the functionality of enzymatic catalysis. Here, we report a coarse-grained triple-basin structure-based model upon ligand binding to describe such multiple-state system by the molecular dynamics simulation. With our model, we have made theoretical predictions that are in good agreement with the experimental measurements. Our results revealed the intrinsic conformational fluctuations between apo and open states without ligand binding. We found that HPPK can switch to the activated holo state upon the ordered binding of the two ligands (ATP and HP). We uncovered the underlying mechanism by which major induced fit and minor population shift pathways coexist upon ligand binding by quantitative flux analysis. Additionally, we pointed out the structural origin for the conformational changes and identified the key residues as well as contact interactions. We further explored the temperature effect on the conformational distributions and pathway weights. It gave strong support that higher temperatures promote population shift, while the induced fit pathway is always the predominant activation route of the HPPK system. These findings will provide significant insights of the mechanisms of the multistate conformational dynamics of HPPK upon ligand binding.

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope.

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Probing single-molecule electron-hole transfer dynamics at a molecule-NiO semiconductor nanocrystalline interface.

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.

Molecular mechanism of multispecific recognition of Calmodulin through conformational changes.

Calmodulin (CaM) is found to have the capability to bind multiple targets. Investigations on the association mechanism of CaM to its targets are crucial for understanding protein-protein binding and recognition. Here, we developed a structure-based model to explore the binding process between CaM and skMLCK binding peptide. We found the cooperation between nonnative electrostatic interaction and nonnative hydrophobic interaction plays an important role in nonspecific recognition between CaM and its target. We also found that the conserved hydrophobic anchors of skMLCK and binding patches of CaM are crucial for the transition from high affinity to high specificity. Furthermore, this association process involves simultaneously both local conformational change of CaM and global conformational changes of the skMLCK binding peptide. We found a landscape with a mixture of the atypical "induced fit," the atypical "conformational selection," and "simultaneously binding-folding," depending on the synchronization of folding and binding. Finally, we extend our discussions on multispecific binding between CaM and its targets. These association characteristics proposed for CaM and skMLCK can provide insights into multispecific binding of CaM.

Revealing Multiple Pathways in T4 Lysozyme Substep Conformational Motions by Single-Molecule Enzymology and Modeling.

Enzyme conformational dynamics play crucial roles in catalytic functions. Obtaining molecular level insights into the conformational transition dynamics of enzyme-substrate complex from the inactive state to the active state is fundamental for understanding enzymatic function and dynamics. Here, we report our progress on the real-time conformational transition dynamics of T4 lysozyme under enzymatic reactions using single-molecule fluorescence resonance energy transfer. The time duration in forming the active enzyme-substrate complex state (ES*) shows distinctive Poisson and non-Poisson statistics, including exponential and nonexponential, convoluted Poisson distributions, and Gaussian-like distributions. These complex dynamic behaviors of T4 lysozyme are in excellent agreement with a Markov dynamic simulation and a transition steps modeling. Specifically, we are able to obtain mechanistic understandings: (1) Transiting from enzyme (E) to ES*, T4 lysozyme hinge-bending conformational changes undergo multiple steps following multiple pathways. In the case of shortest pathway, this transition only requires one elementary transition or reaction step. (2) Substep conformational motions, associating with multiple nuclear coordinates and a common projected FRET-sensitive nuclear coordinate, can give rise to multiple conformational intermediate states. (3) The consequence of the multiple pathways, intermediate states, and nuclear coordinates is the time bunching effect; i.e., time durations of conformational changes tend to bunch in a narrowly distributed time window. The physical picture of multiple intermediate states along with bunching effect suggests that the conformational dynamics of T4 lysozyme shows a complementary characteristic behavior of convoluted enzyme conformation selection and induced-fit dynamics driven by substrate-enzyme interactions.