X-ray structure and mutagenesis analyses of Clostridioides difficile endolysin Ecd09610 glucosaminidase domain.

Clostridioides difficile endolysin (Ecd09610) consists of an unknown domain at its N terminus, followed by two catalytic domains, a glucosaminidase domain and endopeptidase domain. X-ray structure and mutagenesis analyses of the Ecd09610 catalytic domain with glucosaminidase activity (Ecd09610CD53) were performed. Ecd09610CD53 was found to possess an α-bundle-like structure with nine helices, which is well conserved among GH73 family enzymes. The mutagenesis analysis based on X-ray structures showed that Glu405 and Asn470 were essential for enzymatic activity. Ecd09610CD53 may adopt a neighboring-group mechanism for a catalytic reaction in which Glu405 acted as an acid/base catalyst and Asn470 helped to stabilize the oxazolinium ion intermediate. Structural comparisons with the newly identified Clostridium perfringens autolysin catalytic domain (AcpCD) in the P1 form and a zymography analysis demonstrated that AcpCD was 15-fold more active than Ecd09610CD53. The strength of the glucosaminidase activity of the GH73 family appears to be dependent on the depth of the substrate-binding groove.

Biochemical Characterizations of the Putative Amidase Endolysin Ecd18980 Catalytic Domain from Clostridioides difficile.

Clostridioides difficile is the major causative pathogen of pseudomembranous colitis, and novel antimicrobial agents are required for treatment. Phage-derived endolysins exhibiting species-specific lytic activity have potential as novel antimicrobial agents. We surveyed the genome of C. difficile strain 630 and identified a gene encoding an endolysin, Ecd18980, which has an amidase_3 domain at the N-terminus but unknown C-terminal domain. The genes encoding Ecd18980 and its catalytic domain (Ecd18980CD) were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. These purified proteins showed lytic activity against C. difficile. Ecd18980CD showed higher lytic activity than the wild-type enzyme and near-specific lytic activity against C. difficile. This species specificity is thought to depend on substrate cleavage activity rather than binding. We also characterized the biochemical properties of Ecd18980CD, including optimal pH, salt concentration, and thermal stability.

Effects of various sitting and standing postures on arousal and valence.

According to research on the effects of posture on psychological states, high-power poses-with the body spread wide open-lead to high-arousal positive emotions, whereas low-power poses-with the body slumped and constricted-lead to low-arousal negative emotions. However, postures that lead to both high-arousal negative and low-arousal positive emotions have not been investigated yet. Although relative comparisons between postures have been made, the positioning of postures on the two-dimensional coordinates created by arousal and valence has not been clarified. Therefore, the purpose of this study was to explore and clarify which postures lead to the four types of emotions: high-arousal negative, high-arousal positive, low-arousal negative, and low-arousal positive. In Experiment 1, 29 participants (13 men and 16 women) adopted 12 sitting postures for 1 minute each. In Experiment 2, 25 participants (13 men and 12 women) adopted six sitting and six standing postures for 1 minute each. Arousal and valence were measured after each posture, and heart rate was measured during posture maintenance. Arousal and valence after adopting the postures were compared with the neutral arousal and valence. As a result, postures leading to high-arousal negative and low-arousal positive emotions were identified. In addition, postures leading to high-arousal positive emotions, which are the high-power poses, were identified. There were no differences in the magnitude of psychological effects between sitting and standing postures.

Structural and biochemical characterization of Clostridium perfringens pili protein B collagen-binding domains.

Sortase-mediated pili are flexible rod proteins composed of major and minor/tip pilins, playing important roles in the initial adhesion of bacterial cells to host tissues. The pilus shaft is formed by covalent polymerization of major pilins, and the minor/tip pilin is covalently attached to the tip of the shaft involved in adhesion to the host cell. The Gram-positive bacterium Clostridium perfringens has a major pilin, and a minor/tip pilin (CppB) with the collagen-binding motif. Here, we report X-ray structures of CppB collagen-binding domains, collagen-binding assays and mutagenesis analysis, demonstrating that CppB collagen-binding domains adopt an L-shaped structure in open form, and that a small ß-sheet unique to CppB provides a scaffold for a favourable binding site for collagen peptide.

Biochemical Characterizations of the Putative Endolysin Ecd09610 Catalytic Domain from Clostridioides difficile.

Clostridioides difficile is the major pathogen of pseudomembranous colitis, and novel antimicrobial agents are sought after for its treatment. Phage-derived endolysins with species-specific lytic activity have potential as novel antimicrobial agents. We surveyed the genome of C. difficile strain 630 and identified an endolysin gene, Ecd09610, which has an uncharacterized domain at the N-terminus and two catalytic domains that are homologous to glucosaminidase and endopeptidase at the C-terminus. Genes containing the two catalytic domains, the glucosaminidase domain and the endopeptidase domain, were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. The purified domain variants showed lytic activity almost specifically for C. difficile, which has a unique peptide bridge in its peptidoglycan. This species specificity is thought to depend on substrate cleavage activity rather than binding. The domain variants were thermostable, and, notably, the glucosaminidase domain remained active up to 100 °C. In addition, we determined the optimal pH and salt concentrations of these domain variants. Their properties are suitable for formulating a bacteriolytic enzyme as an antimicrobial agent. This lytic enzyme can serve as a scaffold for the construction of high lytic activity mutants with enhanced properties.

Structural and biochemical characterization of the Clostridium perfringens-specific Zn2+-dependent amidase endolysin, Psa, catalytic domain.

Phage-derived endolysins, enzymes that degrade peptidoglycans, have the potential to serve as alternative antimicrobial agents. Psa, which was identified as an endolysin encoded in the genome of Clostridium perfringens st13, was shown to specifically lyse C. perfringens. Psa has an N-terminal catalytic domain that is homologous to the Amidase_2 domain (PF01510), and a novel C-terminal cell wall-binding domain. Here, we determined the X-ray structure of the Psa catalytic domain (Psa-CD) at 1.65 Å resolution. Psa-CD has a typical Amidase_2 domain structure, consisting of a spherical structure with a central ß-sheet surrounded by two α-helix groups. Furthermore, there is a Zn2+ at the center of Psa-CD catalytic reaction site, as well as a unique T-shaped substrate-binding groove consisting of two grooves on the molecule surface. We performed modeling study of the enzyme/substrate complex along with a mutational analysis, and demonstrated that the structure of the substrate-binding groove is closely related to the amidase activity. Furthermore, we proposed a Zn2+-mediated catalytic reaction mechanism for the Amidase_2 family, in which tyrosine constitutes part of the catalytic reaction site.

In vivo Fluorescence Imaging of Extracellular ATP in the Mouse Cerebral Cortex with a Hybrid-type Optical Sensor.

Adenosine 5'-triphosphate (ATP) works as an extracellular signaling molecule for cells in the brain, such as neurons and glia. Cellular communication via release of ATP is involved in a range of processes required for normal brain functions, and aberrant communication is associated with brain disorders. To investigate the mechanisms underlying these cellular processes, various techniques have been developed for the measurement of extracellular ATP. To monitor the dynamics of extracellular ATP signaling with high spatiotemporal resolution, we recently developed a hybrid-type ATP optical sensor (ATPOS) that enables in vivo fluorescence imaging of extracellular ATP dynamics in the brain. ATPOS is synthesized by labeling an ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, with a small-molecular fluorescent dye Cy3. Injection of ATPOS into the cerebral cortex of living mice enables visualization of the wave-like propagation of extracellular ATP release in response to electrical stimulation. The protocol described here should be useful for visualizing ATP signaling in diverse processes involved in intercellular communication in the brain.

A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm.

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme's specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

X-ray structures of Clostridium perfringens sortase C with C-terminal cell wall sorting motif of LPST demonstrate role of subsite for substrate-binding and structural variations of catalytic site.

Pili of Gram-positive bacteria are flexible rod proteins covalently attached to the bacterial cell wall, that play important roles in the initial adhesion of bacterial cells to host tissues and bacterial colonization. Pili are formed by the polymerization of major and minor pilins, catalyzed by class C sortase (SrtC), a family of cysteine transpeptidases. The Gram-positive bacterium Clostridium perfringens has a major pilin (CppA), a minor pilin (CppB), and SrtC (CpSrtC). CpSrtC recognizes the C-terminal cell wall sorting signal motifs with five amino acid residues, LPSTG of CppA and LPETG of CppB, for the polymerization of pili. Here, we report biochemical analysis to detect the formation of Clostridium perfringens pili in vivo, and the X-ray structure of a novel intermolecular substrate-enzyme complex of CpSrtC with a sequence of LPST at the C-terminal site. The results showed that CpSrtC has a subsite for substrate-binding to aid polymerization of pili, and that the catalytic site has structural variations, giving insights into the enzyme catalytic reaction mechanism and affinities for the C-terminal cell wall sorting signal motif sequences.

Structural and biochemical characterizations of the novel autolysin Acd24020 from Clostridioides difficile and its full-function catalytic domain as a lytic enzyme.

Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall-binding (CWB) domains, to be involved in different physiological functions that require bacterial cell wall remodeling. We identified a novel autolysin, Acd24020, from Clostridioides (Clostridium) difficile (C. difficile), with an endopeptidase catalytic domain belonging to the NlpC/P60 family and three bacterial Src-homology 3 domains as CWB domains. The catalytic domain of Acd24020 (Acd24020-CD) exhibited C. difficile-specific lytic activity equivalent to Acd24020, indicating that Acd24020-CD has full-function as a lytic enzyme by itself. To elucidate the specific peptidoglycan-recognition and catalytic reaction mechanisms of Acd24020-CD, biochemical characterization, X-ray structure determination, a modeling study of the enzyme/substrate complex, and mutagenesis analysis were performed. Acd24020-CD has an hourglass-shaped substrate-binding groove across the molecule, which is responsible for recognizing the direct 3-4 cross-linking structure unique to C. difficile peptidoglycan. Based on the X-ray structure and modeling study, we propose a dynamic Cys/His catalyzing mechanism, in which the catalytic Cys299 and His354 residues dynamically change their conformations to complement each step of the enzyme catalytic reaction.

Benzoxazole-Based Metal Complexes to Reverse Multidrug Resistance in Bacteria.

Bacteria often show resistance against antibiotics due to various mechanisms such as the expression of efflux pumps, biofilm formation, or bacterial quorum sensing (QS) controls. For successful therapy, the discovery of alternative agents is crucial. The objective of this study was to evaluate the efflux pump, anti-biofilm, and QS inhibiting, as well as antibacterial effects of 2-trifluoroacetonylbenzoxazole ligands (1-3) and their metal complexes (4-12) in bacteria. The ligand 2 and its Zn(II) complex 5, and furthermore the Cu(II) complex 7 of ligand 1, exerted remarkable antibacterial activity on the Staphylococcus aureus 272123 (MRSA) strain. In the minimum inhibitory concentration (MIC) reduction assay the ligand 3, the Zn(II) complex 5 of ligand 2, and the Cu(II), Ni(II), Mg(II), Fe(III) complexes (7, 8, 9, 12) of ligand 1 enhanced the antibacterial activity of ciprofloxacin in MRSA. An increased ethidium bromide accumulation was detected for ligand 3 in MRSA while the Fe(III) complex 12 of ligand 1 decreased the biofilm formation of the reference S. aureus ATCC 25923 strain. The Zn(II) and Ag(II) complexes (3 and 4) of ligand 1 and ligand 3 inhibited the QS. Based on our results, the ligands and their metal complexes could be potential alternative drugs in the treatment of infectious diseases.

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor.

Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

Biologists often refer to a small molecule called adenosine triphosphate ­ or ATP for short ­ as 'the currency of life'. This molecule carries energy all through the body, and most cells and proteins require ATP to perform their various roles. Nerve cells (also known as neurons) in the brain release ATP when activated, and use this molecule to send signals to other active neurons or other cells in the brain. But ATP can also signal danger in the brain. A molecule derived from ATP is involved in transmitting the pain signals of migraines and severe headaches; and ATP levels can become imbalanced after strokes, when parts of the brain are deprived of blood. Despite its importance, ATP remains difficult to visualize in the body, and monitoring the molecule in the active brain in real time is challenging. To address this issue, Kitajima et al. designed an optical sensor that could monitor ATP in the healthy brain, and was sensitive enough to detect when and where it was released. First, Kitajima et al. made several potential sensors by attaching various fluorescent tags to different locations on a protein that binds ATP. Next each sensor was tested to determine whether it could bind ATP tightly and get bright upon binding. This is important because previous sensors could not detect ATP release in the brains of living animals. To illustrate the new sensors' potential, Kitajima et al. used the sensor to image ATP in the brains of live mice. A 'wave' of ATP was seen spreading through the brain after neurons were stimulated with a small electric pulse, mimicking a sudden migraine or stroke. The results confirm that this new sensor is suitable for imaging how ATP signals in the brain, and it may help resolve the underlying mechanisms of migraines and strokes. This sensor could also be used to understand other cellular process which rely on ATP to carry out their role.

Retrospective analysis of the correlation between tacrolimus concentrations measured in whole blood and variations of blood cell counts in patients undergoing allogeneic haematopoietic stem cell transplantation.

Objective: Tacrolimus is administered to patients undergoing haematopoietic stem cell transplantation (HSCT) as prophylaxis for graft-versus-host disease. As a high blood tacrolimus concentration within a narrow therapeutic range must be maintained after HSCT, therapeutic drug monitoring (TDM) is necessary. We investigated the correlation between blood tacrolimus concentration and blood cell count in HSCT patients to assess how changes in blood cell count affect tacrolimus TDM. Methods: A retrospective analysis was performed for 24 patients who underwent allogeneic HSCT and received tacrolimus. The correlation between variations in blood tacrolimus concentration and blood cell count was evaluated for three consecutive weeks, starting 1 week after HSCT. Results: Variations in blood tacrolimus concentration were significantly correlated with variations in red blood cell (RBC) count, haemoglobin level and haematocrit value, but not with variations in white blood cell or platelet counts. Further, the above variations were significantly correlated in patients undergoing cord blood transplantation and peripheral blood stem cell transplantation, but not in those undergoing bone marrow transplantation. Conclusions: These findings demonstrate that RBC count is associated with variations in blood tacrolimus concentration, with the relevance of this association depending on the source of transfused stem cells. Thus, variations in RBC count might be useful for tacrolimus TDM.

Structures of major pilins in Clostridium perfringens demonstrate dynamic conformational change.

Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric chains in the crystal, yielding a practical model of the pilus fiber structure. The major pilin from strain SM101 adopts a novel bent structure and associates to form a left-handed twist like an antiparallel double helix in the crystal, which is likely to promote bacterial cell-cell interactions. A modeling study showed that pilin with a bent structure interacts favorably with sortase. The major pilin from strain SM101 was considered to be in an equilibrium state between an elongated and a bent structure through dynamic conformational change, which may be involved in pili-mediated colonization and sortase-mediated polymerization of pili.

Movement Modifications Related to Psychological Pressure in a Table Tennis Forehand Task.

Although many studies on choking under pressure used closed skills, such as golf putting, we examined the influence of pressure on movement during a dynamic skill by studying participants' kinematic and kinetic changes during a table tennis forehand task under pressure. Thirty novice table tennis players hit forehand shots toward a target for 135 practice trials and then performed 10 no-pressure and 10 pressure trials. We added psychological pressure by instructing participants they could earn monetary rewards for successful performance and by cancelling accumulated scores for a poor performance. We measured racket head and ball movements as kinematic variables and grip force as a kinetic variable. We also measured state anxiety and heart rate as checks on our manipulation of psychological pressure. In the pressure condition, both state anxiety and heart rate increased significantly ( p < .025), though the pressure level was relatively small. Analysis of kinematic measures revealed that back swing and forward swing were reduced in length; speed of forward swing and ball speed decreased significantly ( p < .008) under pressure. Also, under pressure, ball and racket contact point shifted forward significantly ( p < .008) to reduce the distance between impact and target locations, and performance declined as the ball-landing locations shifted leftward ( p < .007). Grip force showed no significant change. We conclude that, under pressure, movement was modified toward reduced displacement and lower speed in an apparent risk-aversive hitting strategy; these modifications resulted in a performance decrement.

Benzoxazole-based Zn(II) and Cu(II) Complexes Overcome Multidrug-resistance in Cancer.

BACKGROUND/AIM: Multidrug resistance (MDR) represents a significant impediment to successful cancer treatment. In this study, novel metal [Zn(II), Cu(II), Mg(II), Ni(II), Pd(II), and Ag(I)] complexes of 2-trifluoroacetonylbenzoxazole previously synthesized and characterized by our group were tested for their MDR-reversing activity in comparison with the free ligands in L5178Y mouse T-lymphoma (MDR) cells transfected with human ATP-binding cassette sub-family B member 1 (ABCB1; P-glycoprotein) gene. MATERIALS AND METHODS: Cytotoxic and antiproliferative effects of the complexes were assessed by the thiazolyl blue tetrazolium bromide (MTT) method. Modulation of ABCB1 activity was measured by rhodamine 123 accumulation assay using flow cytometry. The apoptosis-inducing activity of some complexes was also tested on the multidrug resistant L5178Y mouse T-lymphoma cells, using the annexin-V/propidium iodide assay. RESULTS: When compared to the free ligand, a remarkable enhancement in MDR reversal and cytotoxic activity was found for the Zn(II) and Cu(II) complexes. The activity of the complexes proved to be up to 29- and 5-fold higher than that of the ligands and the ABCB1 inhibitor verapamil as positive control, respectively. The complexes possessed a remarkable potential to induce apoptosis of MDR cells. CONCLUSION: Our results suggest that the Zn(II) and Cu(II) complexes display significant MDR-reversing activity in a dose-dependent manner and possess strong cytotoxic activity and a remarkable potential to induce apoptosis in MDR L5178Y mouse T-lymphoma cells.

Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents.

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

Synthesis and Antimicrobial Activity of 2-Trifluoroacetonylbenzoxazole Ligands and Their Metal Complexes.

Three 2-fluoroacetonylbenzoxazole ligands 1a-c and their new Zn(II) complexes 2a-c have been synthesized. In addition, syntheses of new metal [Mg(II), Ni(II), Cu(II), Pd(II), and Ag(I)] complexes from 1a have been also described. The molecular and crystal structures of six metal complexes 2b and 2d-h were determined by single-crystal X-ray diffraction analyses. Their antibacterial activities against six Gram-positive and six Gram-negative bacteria were evaluated by minimum inhibitory concentrations (MIC), which were compared with those of appropriate antibiotics and silver nitrate. The results indicate that some metal compounds have more antibacterial effects in comparison with free ligands and have preferred antibacterial activities that may have potential pharmaceutical applications. Noticeably, the Ag(I) complex 2h exhibited low MIC value of 0.7 µM against Pseudomonas aeruginosa, which was even superior to the reference drug, Norfloxacin with that of 1.5 µM. Against P. aeruginosa, 2h is bacteriostatic, exerts the cell surface damage observed by scanning electron microscopy (SEM) and is less likely to develop resistance. The new 2h has been found to display effective antimicrobial activity against a series of bacteria.

A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.

A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.

Real-time observation of the photoionization-induced water rearrangement dynamics in the 5-hydroxyindole-water cluster by time-resolved IR spectroscopy.

Solvation plays an essential role in controlling the mechanism and dynamics of chemical reactions in solution. The present study reveals that changes in the local solute-solvent interaction have a great impact on the timescale of solvent rearrangement dynamics. Time-resolved IR spectroscopy has been applied to a hydration rearrangement reaction in the monohydrated 5-hydroxyindole-water cluster induced by photoionization of the solute molecule. The water molecule changes the stable hydration site from the indolic NH site to the substituent OH site, both of which provide a strongly attractive potential for hydration. The rearrangement time constant amounts to 8 ± 2 ns, and is further slowed down by a factor of more than five at lower excess energy. These rearrangement times are slower by about three orders of magnitude than those reported for related systems where the water molecule is repelled from a repulsive part of the interaction potential toward an attractive well. The excess energy dependence of the time constant is well reproduced by RRKM theory. Differences in the reaction mechanism are discussed on the basis of energy relaxation dynamics.