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Chris M Wood - One of the best experts on this subject based on the ideXlab platform.
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increased gene expression of a Facilitated Diffusion urea transporter in the skin of the african lungfish protopterus annectens during massively elevated post terrestrialization urea excretion
The Journal of Experimental Biology, 2009Co-Authors: Carrie Y C Hung, Fernando Galvez, Chris M WoodAbstract:The full-length cDNA sequence of a putative urea transporter (lfUT) of the Facilitated Diffusion UT-A type has been cloned from the African lungfish Protopterus annectens. The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians ( approximately 65% amino acid homology), followed by mammals and elasmobranchs ( approximately 60%), and then teleosts ( approximately 50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure ('terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12-30 h with rates of 2000-5000 micromol-N kg(-1) h(-1) (versus normal aquatic rates of <130 micromol-N kg(-1) h(-1)) and persisted until 70 h. This appears to occur mainly through the skin. Total 'excess' urea-N excretion amounted to approximately 81,000-91,000 micromol-N kg(-1) over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a Facilitated Diffusion type urea transporter (lfUT) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.
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increased gene expression of a Facilitated Diffusion urea transporter in the skin of the african lungfish protopterus annectens during massively elevated post terrestrialization urea excretion
The Journal of Experimental Biology, 2009Co-Authors: Carrie Y C Hung, Fernando Galvez, Yuen K Ip, Chris M WoodAbstract:The full-length cDNA sequence of a putative urea transporter ( lfUT ) of the Facilitated Diffusion UT-A type has been cloned from the African lungfish Protopterus annectens . The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians (∼65% amino acid homology), followed by mammals and elasmobranchs (∼60%), and then teleosts (∼50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure (`terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12–30 h with rates of 2000–5000 μmol-N kg–1 h–1 (versus normal aquatic rates of<130 μmol-N kg–1 h–1) and persisted until 70 h. This appears to occur mainly through the skin. Total `excess' urea-N excretion amounted to ∼81,000–91,000 μmol-N kg–1 over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a Facilitated Diffusion type urea transporter ( lfUT ) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.
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evidence for Facilitated Diffusion of urea across the gill basolateral membrane of the rainbow trout oncorhynchus mykiss
Biochimica et Biophysica Acta, 2004Co-Authors: Danielle M Mcdonald, Chris M WoodAbstract:Recent in vivo evidence suggests that the mechanism of branchial urea excretion in the ammoniotelic rainbow trout (Oncorhynchus mykiss) is carrier-mediated. Further characterization of this proposed mechanism was achieved by using an in vitro isolated basolateral membrane vesicle (BLMV) preparation in which isolated gill membranes were used to determine a variety of physiological properties of the transporter. BLMV demonstrated two components of urea uptake, a linear component at concentrations up to 17.5 mmol x l(-1) and a saturable component (K(0.5)=0.35+/-0.01 mmol x l(-1); V(max)=0.14+/-0.02 micromol mg protein(-1) h(-1)) with a Hill constant of 1.35+/-0.18 at low, physiologically relevant urea concentrations ( 2 suggesting a protein carrier-mediated process. Combined, this evidence indicates that a Facilitated Diffusion urea transport mechanism is likely present in the basolateral membrane of the rainbow trout gill.
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pulsatile urea excretion in gulf toadfish opsanus beta evidence for activation of a specific Facilitated Diffusion transport system
The Journal of Experimental Biology, 1998Co-Authors: Chris M Wood, Kathlee M Gilmou, S F Perry, Pierre Laure, Patrick J WalshAbstract:When toadfish are made ureotelic by a crowding/confinement protocol, they excrete approximately 90 % of their urea nitrogen (urea-N) production in large, irregular pulses (1-2 pulses per day) from the gill region. We investigated three hypotheses as to the mechanism of pulsatile excretion: (i) the presence of an active reabsorptive 9back-transport9 mechanism that is periodically inhibited to allow urea-N excretion to occur; (ii) the periodic occurrence of a generalized, non-specific increase in gill permeability; and (iii) the presence of a specific Facilitated Diffusion transport system that is periodically activated. Exposure of toadfish during non-pulse periods to treatments designed to block a 9back-transport9 mechanism (Na+-free sea water or the urea analogues 30 mmol l-1 thiourea or 30 mmol l-1 acetamide in the external water) did not stimulate a leakage of urea-N, thereby opposing the first hypothesis. The second hypothesis was opposed by several results. Neither injection of the potent branchial vasodilator L-isoprenaline (10(-5) mol l-1) nor infusion of NH4Cl, the latter at levels known to stimulate urea-N efflux in perfused gills, had any effect on urea-N excretion. Furthermore, during natural pulse events, when the normally very low gill permeability to urea (3x10(-7) cm s-1) increased at least 35-fold, there was no accompanying increase in permeability to either 3H2O (1.5x10(-5) cm s-1) or the paracellular marker [14C]PEG-4000 (10(-8) cm s-1). However [14C]thiourea permeability (1.5x10(-7) cm s-1) increased approximately fivefold, in support of the third hypothesis. Furthermore, when 30 mmol l-1 urea was placed in the external water, a concentration (60 000 micromol-N l-1) approximately three times that of blood (20 000 micromol-N l-1), each efflux pulse event (measured with [14C]urea) was accompanied by a net uptake, such that blood urea-N levels rose rather than fell. A proportional 1:1 relationship between influx per unit external concentration and efflux per unit internal (i.e. plasma) concentration indicated a fully bidirectional transport system. The simultaneous presence of 60 mmol l-1 thiourea in the external water inhibited the influx component by 73 %, further supporting this conclusion. These data, together with recent molecular, morphological and endocrinological evidence, strongly suggest that pulsatile urea-N excretion is caused by the periodic activation of a Facilitated urea transporter in the gills, similar to the vasopressin-regulated urea transporter in the mammalian kidney.
Ralf Metzle - One of the best experts on this subject based on the ideXlab platform.
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Facilitated Diffusion of transcription factor proteins with anomalous bulk Diffusion
Journal of Physical Chemistry B, 2017Co-Authors: Li Liu, Andrey G Cherstvy, Ralf MetzleAbstract:What are the physical laws of the diffusive search of proteins for their specific binding sites on DNA in the presence of the macromolecular crowding in cells? We performed extensive computer simulations to elucidate the protein target search on DNA. The novel feature is the viscoelastic non-Brownian protein bulk Diffusion recently observed experimentally. We examine the influence of the protein–DNA binding affinity and the anomalous Diffusion exponent on the target search time. In all cases an optimal search time is found. The relative contribution of intermittent three-dimensional bulk Diffusion and one-dimensional sliding of proteins along the DNA is quantified. Our results are discussed in the light of recent single molecule tracking experiments, aiming at a better understanding of the influence of anomalous kinetics of proteins on the Facilitated Diffusion mechanism.
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in vivo Facilitated Diffusion model
PLOS ONE, 2013Co-Authors: Maximilia Aue, Ralf MetzleAbstract:Under dilute in vitro conditions transcription factors rapidly locate their target sequence on DNA by using the Facilitated Diffusion mechanism. However, whether this strategy of alternating between three-dimensional bulk Diffusion and one-dimensional sliding along the DNA contour is still beneficial in the crowded interior of cells is highly disputed. Here we use a simple model for the bacterial genome inside the cell and present a semi-analytical model for the in vivo target search of transcription factors within the Facilitated Diffusion framework. Without having to resort to extensive simulations we determine the mean search time of a lac repressor in a living E. coli cell by including parameters deduced from experimental measurements. The results agree very well with experimental findings, and thus the Facilitated Diffusion picture emerges as a quantitative approach to gene regulation in living bacteria cells. Furthermore we see that the search time is not very sensitive to the parameters characterizing the DNA configuration and that the cell seems to operate very close to optimal conditions for target localization. Local searches as implied by the colocalization mechanism are only found to mildly accelerate the mean search time within our model.
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generalized Facilitated Diffusion model for dna binding proteins with search and recognition states
Biophysical Journal, 2012Co-Authors: Maximilia Aue, Ralf MetzleAbstract:Transcription factors (TFs) such as the lac repressor find their target sequence on DNA at remarkably high rates. In the established Berg-von Hippel model for this search process, the TF alternates between three-dimensional Diffusion in the bulk solution and one-dimensional sliding along the DNA chain. To overcome the so-called speed-stability paradox, in similar models the TF was considered as being present in two conformations (search state and recognition state) between which it switches stochastically. Combining both the Facilitated Diffusion model and alternating states, we obtain a generalized model. We explicitly treat bulk excursions for rodlike chains arranged in parallel and consider a simplified model for coiled DNA. Compared to previously considered Facilitated Diffusion models, corresponding to limiting cases of our generalized model, we surprisingly find a reduced target search rate. Moreover, at optimal conditions there is no longer an equipartition between the time spent by the protein on and off the DNA chain.
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Facilitated Diffusion with dna coiling
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Michael A Lomhol, Am Van Den Broek, Svenjamarei J Kalisch, Gijs J L Wuite, Ralf MetzleAbstract:When DNA-binding proteins search for their specific binding site on a DNA molecule they alternate between linear 1-dimensional Diffusion along the DNA molecule, mediated by nonspecific binding, and 3-dimensional volume excursion events between successive dissociation from and rebinding to DNA. If the DNA molecule is kept in a straight configuration, for instance, by optical tweezers, these 3-dimensional excursions may be divided into long volume excursions and short hops along the DNA. These short hops correspond to immediate rebindings after dissociation such that a rebinding event to the DNA occurs at a site that is close to the site of the preceding dissociation. When the DNA molecule is allowed to coil up, immediate rebinding may also lead to so-called intersegmental jumps, i.e., immediate rebindings to a DNA segment that is far away from the unbinding site when measured in the chemical distance along the DNA, but close by in the embedding 3-dimensional space. This effect is made possible by DNA looping. The significance of intersegmental jumps was recently demonstrated in a single DNA optical tweezers setup. Here we present a theoretical approach in which we explicitly take the effect of DNA coiling into account. By including the spatial correlations of the short hops we demonstrate how the Facilitated Diffusion model can be extended to account for intersegmental jumping at varying DNA densities. It is also shown that our approach provides a quantitative interpretation of the experimentally measured enhancement of the target location by DNA-binding proteins.
Carrie Y C Hung - One of the best experts on this subject based on the ideXlab platform.
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increased gene expression of a Facilitated Diffusion urea transporter in the skin of the african lungfish protopterus annectens during massively elevated post terrestrialization urea excretion
The Journal of Experimental Biology, 2009Co-Authors: Carrie Y C Hung, Fernando Galvez, Yuen K Ip, Chris M WoodAbstract:The full-length cDNA sequence of a putative urea transporter ( lfUT ) of the Facilitated Diffusion UT-A type has been cloned from the African lungfish Protopterus annectens . The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians (∼65% amino acid homology), followed by mammals and elasmobranchs (∼60%), and then teleosts (∼50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure (`terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12–30 h with rates of 2000–5000 μmol-N kg–1 h–1 (versus normal aquatic rates of<130 μmol-N kg–1 h–1) and persisted until 70 h. This appears to occur mainly through the skin. Total `excess' urea-N excretion amounted to ∼81,000–91,000 μmol-N kg–1 over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a Facilitated Diffusion type urea transporter ( lfUT ) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.
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increased gene expression of a Facilitated Diffusion urea transporter in the skin of the african lungfish protopterus annectens during massively elevated post terrestrialization urea excretion
The Journal of Experimental Biology, 2009Co-Authors: Carrie Y C Hung, Fernando Galvez, Chris M WoodAbstract:The full-length cDNA sequence of a putative urea transporter (lfUT) of the Facilitated Diffusion UT-A type has been cloned from the African lungfish Protopterus annectens. The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians ( approximately 65% amino acid homology), followed by mammals and elasmobranchs ( approximately 60%), and then teleosts ( approximately 50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure ('terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12-30 h with rates of 2000-5000 micromol-N kg(-1) h(-1) (versus normal aquatic rates of <130 micromol-N kg(-1) h(-1)) and persisted until 70 h. This appears to occur mainly through the skin. Total 'excess' urea-N excretion amounted to approximately 81,000-91,000 micromol-N kg(-1) over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a Facilitated Diffusion type urea transporter (lfUT) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.
Fernando Galvez - One of the best experts on this subject based on the ideXlab platform.
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increased gene expression of a Facilitated Diffusion urea transporter in the skin of the african lungfish protopterus annectens during massively elevated post terrestrialization urea excretion
The Journal of Experimental Biology, 2009Co-Authors: Carrie Y C Hung, Fernando Galvez, Yuen K Ip, Chris M WoodAbstract:The full-length cDNA sequence of a putative urea transporter ( lfUT ) of the Facilitated Diffusion UT-A type has been cloned from the African lungfish Protopterus annectens . The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians (∼65% amino acid homology), followed by mammals and elasmobranchs (∼60%), and then teleosts (∼50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure (`terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12–30 h with rates of 2000–5000 μmol-N kg–1 h–1 (versus normal aquatic rates of<130 μmol-N kg–1 h–1) and persisted until 70 h. This appears to occur mainly through the skin. Total `excess' urea-N excretion amounted to ∼81,000–91,000 μmol-N kg–1 over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a Facilitated Diffusion type urea transporter ( lfUT ) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.
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increased gene expression of a Facilitated Diffusion urea transporter in the skin of the african lungfish protopterus annectens during massively elevated post terrestrialization urea excretion
The Journal of Experimental Biology, 2009Co-Authors: Carrie Y C Hung, Fernando Galvez, Chris M WoodAbstract:The full-length cDNA sequence of a putative urea transporter (lfUT) of the Facilitated Diffusion UT-A type has been cloned from the African lungfish Protopterus annectens. The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians ( approximately 65% amino acid homology), followed by mammals and elasmobranchs ( approximately 60%), and then teleosts ( approximately 50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure ('terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12-30 h with rates of 2000-5000 micromol-N kg(-1) h(-1) (versus normal aquatic rates of <130 micromol-N kg(-1) h(-1)) and persisted until 70 h. This appears to occur mainly through the skin. Total 'excess' urea-N excretion amounted to approximately 81,000-91,000 micromol-N kg(-1) over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a Facilitated Diffusion type urea transporter (lfUT) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.
Silvio Cavalcanti - One of the best experts on this subject based on the ideXlab platform.
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insights into the sliding movement of the lac repressor nonspecifically bound to dna
Journal of Physical Chemistry B, 2010Co-Authors: Simone Furini, Carmen Domene, Silvio CavalcantiAbstract:: The Lac repressor finds its DNA binding sequences with an association rate 2 orders of magnitude higher than what is expected for a random diffusive process. This experimental data stimulated numerous theoretical and experimental studies, which led to the Facilitated Diffusion model. In Facilitated Diffusion, the Lac repressor binds nonspecifically to DNA. This nonspecific binding is followed by an exploration of the DNA molecule in a reduced space. Single-molecule imaging confirmed that the Lac repressor may move along the DNA molecule; however, it is still under debate whether the LacI movement proceeds through sliding, with a continuous close contact between the protein and DNA, or through hopping between adjacent binding sites. We have investigated the one-dimensional sliding movement of the Lac repressor along nonspecific DNA by full-atomistic molecular dynamics simulations and free-energy calculations based on the umbrella sampling technique. The computed free-energy profile along a helical trajectory was periodic, with periodicity equal to the distance between successive nucleotides and an energy barrier between successive minima of 8.7 +/- 0.7 kcal/mol. The results from the molecular simulations were subsequently used in a Langevin dynamics framework to estimate the Diffusion coefficient of the Lac repressor sliding along nonspecific DNA. The computed Diffusion coefficient is close to the lower limit of the experimental range.
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a multiscale model to analyze the sliding movement of repressor proteins on dna
Biophysical Journal, 2010Co-Authors: Simone Furini, Carmen Domene, Silvio CavalcantiAbstract:Repressor proteins (RP) regulate gene transcriptions by binding to target sequences, named operator sites, on the DNA molecule. Association rates higher than the Diffusion limit were measured in several RP. These experimental data led to the Facilitated Diffusion model. Facilitated Diffusion requires nonspecific binding of the RP to the DNA. Then, the searching for the target sequence proceeds in a reduced search space. In agreement with this model, a structure of the RP LacI bound to nonspecific DNA was revealed by NMR, and one-dimensional movements of the same protein along DNA were observed by single molecule imaging. Single molecule imaging cannot provide a molecular description of how the movement occurs at the molecular level, and two hypotheses were formulated: i) sliding of the RP, in continuous contact with the DNA major grove; ii) hopping of the RP between adjacent binding sites. The continuous contact between the protein and the DNA major grove can result only from a helical trajectory of the RP around the DNA molecule. We simulated the sliding motion of the LacI protein along this helical trajectory by a multiscale model than integrates data from molecular dynamics (MD) simulations in stochastic dynamics. The multiscale approach was necessary to extend the timescale accessible by brute-force MD, and simulate dynamics on the millisecond timescale. MD simulations were used to compute the local Diffusion coefficient and the potential of mean force for the sliding movement. These data were then used in the stochastic simulations, to simulate the dynamics on a millisecond time scale, and identify the characteristics of the hypothetic sliding motion. Since the parameters of the stochastic equation were computed by MD simulations, the multiscale model is strictly based on the microscopic characteristics of the molecular system.
