Rebinding Dynamics Research Articles

Mapping fast DNA polymerase exchange during replication

Despite extensive studies on DNA replication, the exchange mechanisms of DNA polymerase during replication remain unclear. Existing models propose that this exchange is facilitated by protein partners like helicase. Here we present data, employing a combination of mechanical DNA manipulation and single fluorescent protein observation, that reveal DNA polymerase undergoing rapid and autonomous exchange during replication not coordinated by other proteins. The DNA polymerase shows fast unbinding and rebinding dynamics, displaying a preference for either exonuclease or polymerase activity, or pausing events, during each brief binding event. We also observed a ‘memory effect’ in DNA polymerase rebinding, i.e., the enzyme tends to preserve its prior activity upon reassociation. This effect, potentially linked to the ssDNA/dsDNA junction’s conformation, might play a role in regulating binding preference enabling high processivity amidst rapid protein exchange. Taken together, our findings support an autonomous replication model that includes rapid protein exchange, burst of activity, and a ‘memory effect’ while moving processively forward.

A new method to experimentally quantify dynamics of initial protein–protein interactions

Cells run on initiation of protein-protein interactions, which are dynamically tuned spatially and temporally to modulate cellular events. This tuning can be physical, such as attaching the protein to a cargo or protein complex, thereby altering its diffusive properties, or modulating the distance between protein pairs, or chemical, by altering the proteins’ conformations (e.g., nucleotide binding state of an enzyme, post-translational modification of a protein, etc.). Because a dynamic and changing subset of proteins in the cell could be in any specific state, ensemble measurements are not ideal—to untangle which of the factors are important, and how, we need single-molecule measurements. Experimentally, until now we have not had good tools to precisely measure initiation of such protein-protein interactions at the single-molecule level. Here, we develop a new method to measure dynamics of initial protein-protein interactions, allowing measurement of how properties such as the distance between proteins, and their tethered length can modulate the rate of interactions. In addition to precise measurement distance dependent motor-MT rebinding dynamics, we demonstrate the use of a dithered optical trap to measure dynamic motor-MT interactions and further discuss the possibilities of this technique being applicable to other systems.

Effect of Single-Point Mutations on Nitric Oxide Rebinding and the Thermodynamic Stability of Myoglobin.

The effect of single amino acid mutations on the rebinding dynamics of nitrogen monoxide (NO) to myoglobin is investigated using reactive molecular dynamics simulations. In particular, mutations of residues surrounding the heme-active site (Leu29, His64, Val68) were considered. Consistent with experiments, all mutations studied here have a significant effect on the kinetics of the NO-rebinding process, which consists of a rapid (several 10 ps) and a slow (100s of ps) time scale. For all modifications considered, the time scales and rebinding fractions agree to within a few percents with results from experiments by adjusting one single, physically meaningful, conformationally averaged quantity: the asymptotic energy separation between the NO-bound (2A) and photodissociated (4A) states. It is furthermore shown that the thermodynamic stability of wild-type versus mutant Mb for the ligand-free and ligand-bound variants of the protein can be described by the same computational model. Therefore, ligand kinetics and thermodynamics are related in a direct fashion akin to Φ-value analysis, which establishes a relationship between protein folding rates and thermal stability of proteins.

Electrochemical sensor based on molecularly imprinted composite membrane of poly(o-aminothiophenol) with gold nanoparticles for sensitive determination of herbicide simazine in environmental samples

A novel electrochemical sensor based on molecularly imprinted polymer membranes (MIPM) as biomimetic molecular recognition element involved in o-aminothiophenol functionalized Au nanoparticles (ATP@AuNPs) modified gold electrode was constructed for sensitive and selective detection of herbicide simazine (SMZ). The nano-scaled MIPM, with high specific surface area, was prepared by self-assembly of o-aminothiophenol (ATP) and electrodeposition of ATP@AuNPs in the presence of template SMZ. Cathodic current of SMZ was measured by cyclic voltammetry and the results exhibited that the proposed sensor possess a high electrocatalytic activity at a negative potential and a fast rebinding dynamics towards the reduction of SMZ in 0.01M H2SO4 solution (pH 1.7). Linear dependency of peak current on SMZ concentrations was observed from 0.03 to 140μM and detection limit was estimated to be 0.013μM (3S/N). The enhancement of sensitivity was attributed to the presence of gold nanoparticles (AuNPs) which decreased the electron-transfer impedance and increased imprinting sites, lead to the superior enrichment effect of the trace amount of SMZ in aqueous solvent. The developed SMZ imprinted sensor exhibited excellent long-term stability and acceptable repeatability. In addition, the proposed method was successfully applied to measure SMZ in several real samples with the spiked recoveries changing from 91.4% to 96.8%, showing a promising potential in practical application.

Based on magnetic graphene oxide highly sensitive and selective imprinted sensor for determination of sunset yellow

A new imprinted material based on β-cyclodextrin/ionic liquid/gold nanoparticles functionalized magnetic graphene oxide has been successfully synthesized and modified to the glassy carbon electrode surface to constructed imprinted electrochemical sensor to detect sunset yellow. The sensitivity and electrochemical response of the electrode can be improved by nanomaterials. The surface morphology and crystal structure of the hybrid nanomaterial has been characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy. The electrochemical behaviors of the hybrid nanomaterials based sensor were evaluated through cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimized conditions, the proposed electrochemical sensor showed a fast rebinding dynamics, which was successfully applied to sunset yellow detection with a wide linear range from 5.0×10−9 to 2.0×10−6molL−1 and a detection limit of 2.0×10−9molL−1. The electrochemical sensor has been successfully applied in the determination of SY in spiked water samples, mirinda drink and minute maid, and the recoveries for the standards added are 97–105%.

An ionic liquid-modified graphene based molecular imprinting electrochemical sensor for sensitive detection of bovine hemoglobin

A novel kind of molecular imprinted polymers based on ionic liquid-functionalized graphene (MIPs/IL/GR) was prepared by electro-polymerization, which was applied as a molecular recognition element to modify glassy carbon electrode (GCE) to construct an electrochemical sensor (MIPs/IL/GR/GCE) for sensitive detection of bovine hemoglobin (BHb). The fabrication conditions that affect the performance of the imprinted sensor, such as pyrrole concentration, scan cycles and scan rates, have been discussed. Under the optimized conditions, the prepared molecular imprinting electrochemical sensor showed a fast rebinding dynamics, which was successfully applied to BHb detection with a wide linear range from 1.0×10−10 to 1.0×10−3g/L (R=0.998) and a detection limit of 3.09×10−11g/L. Moreover, the fabricated sensor possessed a good selectivity and stability, providing a promising tool for immunoassays and clinical applications.

Sensitive and selective electrochemical determination of quinoxaline-2-carboxylic acid based on bilayer of novel poly(pyrrole) functional composite using one-step electro-polymerization and molecularly imprinted poly(o-phenylenediamine)

A facile and efficient molecularly imprinted polymer (MIP) recognition element of electrochemical sensor was fabricated by directly electro-polymerizing monomer o-phenylenediamine (oPD) in the presence of template quinoxaline-2-carboxylic acid (QCA), based on one-step controllable electrochemical modification of poly(pyrrole)-graphene oxide-binuclear phthalocyanine cobalt (II) sulphonate (PPY-GO-BiCoPc) functional composite on glassy carbon electrode (GCE). The MIP film coated on PPY-GO-BiCoPc functional composite decorated GCE (MIP/PPY-GO-BiCoPc/GCE) was presented for the first time. The synergistic effect and electro-catalytic activity toward QCA redox of PPY-GO-BiCoPc functional composite were discussed using various contrast tests. Also, the effect of experimental variables on the current response such as, electro-polymerization cycles, template/monomer ratio, elution condition for template removal, pH of the supporting electrolyte and accumulation time, were investigated in detail. Under the optimized conditions, the proposed MIP sensor possessed a fast rebinding dynamics and an excellent recognition capacity to QCA, while the anodic current response of square wave voltammetry (SWV) was well-proportional to the concentration of QCA in the range of 1.0×10−8–1.0×10−4 and 1.0×10−4–5.0×10−4molL−1 with a low detection limit of 2.1nmolL−1. The established sensor was applied successfully to determine QCA in commercial pork and chicken muscle samples with acceptable recoveries (91.6–98.2%) and satisfactory precision (1.9–3.5% of SD), demonstrating a promising feature for applying the MIP sensor to the measurement of QCA in real samples.

“Strange Kinetics” in the Temperature Dependence of Methionine Ligand Rebinding Dynamics in Cytochrome c

The temperature dependence of methionine ligand dissociation and rebinding dynamics in cytochrome c in aqueous solution has been studied using classical molecular dynamics simulation. Results are compared with previous study of rebinding dynamics at 300 K in water in order to understand how the change of protein environment and the underlying protein energy landscape influence the dynamics. Rebinding dynamics at 77, 180, and 300 K exhibits changes in both time scale and mechanism as the protein and solvent undergo a dynamic "glass transition". At each temperature, the rebinding dynamics yields a subset of trajectories that undergo fast rebinding as well as a subset of trajectories that undergo slower rebinding. At 300 K in water, both a fast (4.0 ps) and slow (14.6 ps) rebinding is observed. While fast rebinding occurs from a "downward" (heme pointing) substate of the methionine, the slow rebinding involves interconversion between an "upward" substate, from which rebinding cannot occur, and the downward substate. At lower temperatures (77 and 180 K), the upward dissociated substate was not observed due to the high barrier imposed by the "frozen" protein structure. However, a slow rebinding phase is observed at both 77 and 180 K and is associated with a process of trapping in downward but "binding forbidden" substates with subsequent slow dynamical conversion to "binding competent" substates from which rebinding is relatively rapid. Distinctive rebinding dynamics at 77 and 180 K suggest that different rebinding time scales are predetermined by the protein and solvent structural arrangement prior to photodissociation, which causes either fast rebinding (about 2 ps) or slow (>50 ps) rebinding. Suggestions for future experiments to further probe the role of dynamic heterogeneity in the kinetics of methionine ligand binding in cytochrome c protein are proposed.

Geminate rebinding dynamics of nitric oxide to ferric hemoglobin in D2O solution

Femtosecond mid-infrared (mid-IR) spectroscopy was used to probe geminate rebinding (GR) dynamics of photo-released nitric oxide (NO) to ferric hemoglobin (Hb(III)) in D2O solution at room temperature. Time-resolved vibrational spectra exhibit two overlapping NO bands for NO-bound Hb(III) (Hb(III)NO), a major band at 1925 cm(-1) (89%) and a minor one at 1905 cm(-1) (11%), suggesting that Hb(III)NO has at least two conformational substates. Both bands decay nonexponentially, each with a different time scale, and the decays are described by a stretched exponential function; the major band's decay is described by 0.96 exp(-t/40 ps)(0.86) + 0.04 and the minor band's decay is described by exp(-t/85 ps)(0.75). These decays arise mainly from the GR of the photo-released NO to Hb(III), indicating that the bound state's conformer influences the NO binding. In particular, the His64 residue, known to have inward conformation in the major band and outward conformation in the minor band, plays a significant role in controlling the binding of NO to Hb(III). The GR of NO to ferric Hb is slower than that to ferrous Hb, which shows fast and efficient GR due to the high reactivity of NO to the heme Fe(ii). The slower GR of NO to Hb(III) may be caused by the lower reactivity of NO to the heme Fe(iii).

Dynamics of Geminate Rebinding of CO to Cytochrome c in Guanidine HCl Probed by Femtosecond Vibrational Spectroscopy

Femtosecond vibrational spectroscopy was used to probe the rebinding dynamics of CO to cytochrome c (Cytc) in 1.8 and 7 M guanidine HCl (GdnHCl) after photodeligation of the corresponding CO-bound protein in D2O buffer (pD = 7.4) at 283 K. Geminate rebinding (GR) dynamics of CO to the folded Cytc in 1.8 M GdnHCl (nCytc) is similar to that to chemically modified cytochrome c (cCytc), suggesting that the overall conformations of nCytcCO and cCytcCO are similar. About 86% of the dissociated CO molecules were geminately rebound to nCytc nonexponentially within 1 ns. The efficient GR of CO to the folded Cytc can be attributed to the organized protein matrix near the active site of nCytc that provides an efficient trap for the diffusing CO ligand after photodissociation. Although the concentration of nCytc did not affect its GR yield of CO, GR yield of CO to the unfolded Cytc in 7 M GdnHCl (uCytc) increased from 5 to 30% as the protein concentration increased from 0.3 to 9 mM. Time-resolved spectra of the (13)CO dissociated from both 9 mM nCytc(13)CO and 9 mM uCytc(13)CO showed a growing band with a peak at 2090 cm(-1) on the picosecond time scale, which was assigned to (13)CO in D2O solvent. At 1 ns, the fraction of the CO band in the solvent was about 10% of the nascent photodeligated protein in nCytc and more than 50% in the concentrated uCytc. Whereas a small opening in the active site of nCytc is responsible for the ultrafast escape of CO to solution in the folded protein, a large fraction of the CO escape to the solvent in uCytc results from the denatured structure of the active site in the unfolded protein. The spectrum of the CO dissociated from the concentrated uCytcCO contained a band that decayed as efficiently as that for the folded protein, suggesting that some fraction of uCytcCO might form aggregates even in 7 M denaturant, such that the aggregate acts as an efficient trap for the diffusing CO after deligation. No hint of precipitate in the concentrated uCytcCO and protein refolding upon dilution of the GdnHCl indicate that the aggregate does not grow continuously but remains as a soluble oligomer. The delayed appearance of the solvated CO and the inefficient GR of CO in uCytcCO suggest that the monomeric unfolded CytcCO so loosely arranged that the protein matrix cannot trap CO efficiently but the bound CO is still buried within hydrophobic residues even under the harsh denaturation condition.

Real-time tracking of CO migration and binding in the α and β subunits of human hemoglobin via 150-ps time-resolved Laue crystallography

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100ps to 10μs. The proximity of the B site in the β subunit is about 0.25Å closer to its A binding site, and its kBA rebinding rate (∼300μs−1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the β subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2.

Dynamics of Geminate Rebinding of NO with Cytochrome c in Aqueous Solution Using Femtosecond Vibrational Spectroscopy

Using femtosecond vibrational spectroscopy, we investigated the rebinding dynamics of NO to cytochrome c (Cytc) and a model heme, microperoxidase-8 (Mp), after photodeligation of CytcNO in D(2)O solution and MpNO in an 81% glycerol/water (v/v) mixture at room temperature. Whereas the stretching mode of the NO band in MpNO was described by a Gaussian centered at 1653 cm(-1) with a full width at half-maximum (fwhm) of 41 cm(-1), that in CytcNO revealed an asymmetric structured band that peaked at 1619 cm(-1) with an fwhm of about 27 cm(-1). The structured NO band in CytcNO was well described by the sum of three Gaussians, and its shape did not evolve with time but its amplitude decayed exponentially with a time constant of 7 ± 1 ps. The transient NO band in MpNO also decayed exponentially with a time constant of 8 ± 1 ps. Rebinding of NO to Cytc was slightly faster than that of NO to Mp and was almost complete by 30 ps, which was much faster than the rebinding of NO to myoglobin (Mb). When the deligated NO was constrained near the Fe atom either by a viscous solvent or by the protein matrix, it rebound to heme Fe much faster than CO, suggesting that NO has a higher propensity for binding to heme Fe and the high reactivity governed the rebinding kinetics. Moreover, the faster ligand rebinding in Cytc than in Mb suggests that Cytc does not have a primary docking site (PDS)-like structure found in Mb that suppresses rebinding by restraining ligand motion and the PDS can also hold the deligated NO in a manner that impedes NO rebinding; however, due to higher NO reactivity with heme Fe, the impediment is not as efficient as for CO.

Molecularly imprinted polymers based electrochemical sensor for bovine hemoglobin recognition

A simple and efficient molecularly imprinted polymers (MIPs) based electrochemical sensor were proposed and prepared by electropolymerization of pyrrole in the presence of bovine hemoglobin (BHb) in an aqueous solution. The fabrication process of the sensor was characterized by differential pulse voltammetry and electrochemical impedance spectroscopy, in which [Fe(CN)6]3−/[Fe(CN)6]4− was used as an electrochemical active probe. The influence factors including electropolymerization cycles, scan rate, the concentration of pyrrole, and the extraction conditions were investigated in detailed. Under the optimized conditions, the experimental results showed that the MIPs based sensor possessed a fast rebinding dynamics and an excellent recognition capacity to BHb, compared to the other non-template proteins. Moreover, the prepared sensor also exhibited a dependent relationship between the concentration of template protein and peak current of [Fe(CN)6]3−/[Fe(CN)6]4−.

Reversible Hydrogen Bond Network Dynamics: Molecular Dynamics Simulations of Calix[4]arene-Catenanes

We present detailed molecular dynamics (MD) simulations of mechanically interlocked calix[4]arene-catenanes under external force. Single-molecule force spectroscopy experiments revealed that the separation of dimers with two aliphatic loops results in reversible hydrogen bond breakage through an intermediate in a triple-well potential, while the tetra-loop species separates in a one-step manner (Janke, M.; et al. Nat. Nanotechnol. 2009, 4, 225). MD simulations show that calix[4]arenes interlocked by four loops (1) display a complete restructuring of the hydrogen bond network under mechanical force. All hydrogen bonds of the closed structure open, and new ones are formed in the extended structure. For small loading rates, we found reversible rejoining of the hydrogen bond network, while the rebinding ability diminishes with increasing pulling velocity, demonstrating the feasibility of MD simulations to capture also rebinding dynamics. Calix[4]arene dimers with two longer loops (2) under external force display more intricate physics because the elongation proceeds in a two-step transition from a compact structure to an open one, in which complete dissociation into calix[4]arene monomers is only prevented by the mechanical locking of the loops. We present a detailed analysis of hydrogen bond breakage and show that the transition from the closed to the intermediate structure is very similar to the transition from the closed to the open structure in the tetra-loop case. The stability of the intermediate is explained in terms of the closed hydrogen bonds, which are only broken when the transition to the open structure is enforced.

Structurally Selective Geminate Rebinding Dynamics of Solvent-Caged Radicals Studied with Nonequilibrium Infrared Echo Spectroscopy

Cyclopentadienylmolybdenum(II) tricarbonyl dimer exists in two different equilibrium conformations: trans and gauche. Ultrafast photoexcitation in the ultraviolet cleaves the Mo-Mo bond, permitting observation of the subsequent geminate rebinding reaction (t(rebinding) = 31.6 ps) by monitoring infrared bleach recoveries at frequencies corresponding to the CO stretches of the trans and gauche isomers, the time-resolved measurements revealed that the monomers rebind in the trans configuration only. Further insight into the rebinding reaction was obtained by mapping the full potential energy surface along the reaction coordinate using electronic-structure methods.

Rebinding Dynamics of CO Following Photodissociation of 4.0 M Guanidine HCl-Denatured Carbonmonoxyhemoglobin

13 CO. Geminate rebinding of CO to the denatured Hb was accelerated more than 1000 times, suggesting that the native structure of the Hb is required to suppress efficient geminate rebinding of CO, as is the case in Mb. The geminate yield and rate for CO rebinding are almost the same in both the denatured Hb and Mb. Similarity in the equilibrium spectrum and rebinding dynamics of CO indicates that the state of the denatured Hb is very similar to that of the denatured Mb. In the denatured Hb, quaternary contact of the protein is likely severed, with the denatured protein existing as an independent subunit much like Mb. Mb, an oxygen storage protein, is a monomeric protein that contains a single heme group as the active ligand-binding site of the protein. Hb, an oxygen transport protein, is a tetrameric protein, each unit of which contains a heme group, with the global tertiary structure of each unit similar to that of Mb. Hb exhibits cooperative binding of ligands essential to efficient transport of O2. 2 The cooperativity of Hb is accompanied by a substantial change in its quaternary structure. Whereas the peptide sequence of Mb is different from that of Hb, amino acid residues near the heme in both proteins are highly conserved. The residues surrounding the bound ligand are invariant in both proteins, except for isoleucine 107 in Mb that is replaced with leucine 107 in Hb. The rebinding of CO to Mb and Hb after photolysis are most often used to study the relationships between protein structure, function, and dynamics. Though the rebinding dynamics of the ligands to both proteins are similar in general, they are different in detail. Whereas cooperative binding of the ligand to Hb makes its behavior difficult to compare directly to the binding of Mb, the difference is often attributed to arise from the quaternary structure of Hb. The dynamics and structural characterization of denatured proteins can be useful in understanding protein folding as well as various biological phenomena such as regulation of the cellular activity and onset of neurodegenerative disease. 3

Dynamics of Ultrafast Rebinding of CO to Carboxymethyl Cytochrome c

Rebinding dynamics of CO to carboxymethyl cytochrome c (Ccytc), a chemically modified cytochrome c to bind ligands in its ferrous form, in D(2)O solution at 283 K after photodeligation, was investigated using femtosecond vibrational spectroscopy. The stretching mode of CO bound to the protein shows four stretching bands near 1962 cm(-1). Time-resolved spectra of the bound CO revealed a slight band-position-dependent rebinding kinetics, suggesting that the geminate rebinding of CO depends on the conformation of the protein. The overall rebinding kinetics of CO to Ccytc was more than 1000 times faster than that to myoglobin (Mb), a ligand-binding protein, and is also faster than a model heme, microperoxidase-8 in viscous solvent. The efficient rebinding of CO to Ccytc was attributed to the longer retention of the dissociated CO near the active binding site by the organized protein matrix of Ccytc. The spectra of the dissociated CO reveal a fast-growing band in the picosecond time scale that is assigned to CO in D(2)O solvent. The ultrafast CO escape to bulk solution is consistent with its 3D structure showing a sizable opening in the active site. It appears that most of the dissociated CO rebinds within 1 ns, except for those that escape to the bulk solution through the opening. The CO rebinding in Ccytc indicates that the primary heme pocket in Mb, located near the active site and holding the dissociated ligand for longer than tens of nanoseconds, has a specific structure to suppress CO rebinding.

Diffusive dynamics on multidimensional rough free energy surfaces

The dynamics of processes relevant to chemistry and biophysics on rough free energy landscapes is investigated using a recently developed algorithm to solve the Smoluchowski equation. Two different processes are considered: ligand rebinding in MbCO and protein folding. For the rebinding dynamics of carbon monoxide (CO) to native myoglobin (Mb) from locations around the active site, the two-dimensional free energy surface (FES) is constructed using extensive molecular dynamics simulations. The surface describes the minima in the A state (bound MbCO), CO in the distal pocket and in the Xe4 pocket, and the transitions between these states and allows to study the diffusion of CO in detail. For the folding dynamics of protein G, a previously determined two-dimensional FES was available. To follow the diffusive dynamics on these rough free energy surfaces, the Smoluchowski equation is solved using the recently developed hierarchical discrete approximation method. From the relaxation of the initial nonequilibrium distribution, experimentally accessible quantities such as the rebinding time for CO or the folding time for protein G can be calculated. It is found that the free energy barrier for CO in the Xe4 pocket and in the distal pocket (B state) closer to the heme iron is approximately 6 kcal/mol which is considerably larger than the inner barrier which separates the bound state and the B state. For the folding of protein G, a barrier of approximately 10 kcal/mol between the unfolded and the folded state is consistent with folding times of the order of milliseconds.

Studying Reactive Processes with Classical Dynamics: Rebinding Dynamics in MbNO

A new surface-crossing algorithm suitable for describing bond-breaking and bond-forming processes in molecular dynamics simulations is presented. The method is formulated for two intersecting potential energy manifolds which dissociate to different adiabatic states. During simulations, crossings are detected by monitoring an energy criterion. If fulfilled, the two manifolds are mixed over a finite number of time steps, after which the system is propagated on the second adiabat and the crossing is carried out with probability one. The algorithm is extensively tested (almost 0.5 μs of total simulation time) for the rebinding of NO to myoglobin. The unbound surface (Fe···NO) is represented using a standard force field, whereas the bound surface (Fe–NO) is described by an ab initio potential energy surface. The rebinding is found to be nonexponential in time, in agreement with experimental studies, and can be described using two time constants. Depending on the asymptotic energy separation between the manifolds, the short rebinding timescale is between 1 and 9 ps, whereas the longer timescale is about an order of magnitude larger. NO molecules which do not rebind within 1 ns are typically found in the Xenon-4 pocket, indicating the high affinity of NO to this region in the protein.

Free-Energy Barriers in MbCO Rebinding

The rebinding of CO to myoglobin (Mb) from locations around the active site is studied using a combination of molecular dynamics and stochastic simulations for native and L29F mutant Mb. The interaction between the dissociated ligand and the protein environment is described by the recently developed fluctuating three-point charge model for the CO molecule. Umbrella sampling along trajectories, previously found to sample the binding site (B) and the Xe4 pocket, is used to construct free-energy profiles for the ligand escape. On the basis of the Smoluchowski equation, the relaxation of different initial population distributions is followed in space and time. For native Mb at room temperature, the calculated rebinding times are in good agreement with experimental values and give an inner barrier of 4.3 kcal/mol between the docking site B (Mb...CO) and the A state (bound MbCO), compared to an effective barrier, Heff, of 4.5 kcal/mol and barriers into the majority conformation A1 and the minority conformation A3 of 2.4 and 4.3 kcal/mol, respectively. In the case of the L29F mutant, the free-energy surface is flatter and the dynamics is much more rapid. As was found in experiment, escape to the Xe4 pocket is facile for L29F whereas, for native Mb, the barriers to this site are larger. At lower temperatures, the rebinding dynamics is delayed by orders of magnitude also due to increased barriers between the docking sites.