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Y Engelborghs

Publications and source records attributed to Y Engelborghs.

At least 37 records · Page 2Linked to original sources
PubMed1999

Dynamics of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White.

Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated alpha 1-acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated alpha 1-acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated alpha 1-acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.

Benzenesulfonates↗
PubMed1999

Fluorescence quenching in the DsbA protein from Escherichia coli: complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan.

The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and is required for efficient disulfide bond formation in the bacterial periplasm. DsbA contains two tryptophans: W76 and W126. The fluorescence of W76 changes upon reduction of the disulfide bridge, as analyzed previously (Hennecke et al., Biochemistry 1997;36:6391-6400). The fluorescence of W126 is highly quenched. The only two potential side chain quenchers are Q74 and N127, and these were replaced by alanine, resulting in a threefold increase in fluorescence intensity. The fluorescence intensity increase is not due to the removal of dynamic quenchers but to an increase in the population with the longest lifetime. In this report, the possibility of a change in the conformation of W126 is investigated theoretically by using molecular mechanics and dynamic simulations and experimentally by using a reaction with N-bromosuccinimide. This reacts preferably with the most exposed microstate of tryptophan, which is responsible for the longest lifetime. The simulations and the experimental results reveal that the amino acid replacements allow W126 to increase the population of its antiperpendicular conformation. The selectivity of the N-bromosuccinimide reaction allows the visualization of the reshuffling kinetics at exhausting reagent concentration. To the authors' knowledge, this is the first time that the kinetics of Trp population reshuffling have been measured.

Crystallography, X-Ray↗
PubMed1999

Time resolved fluorescence and phosphorescence properties of the individual tryptophan residues of barnase: evidence for protein-protein interactions.

Steady-state and time-resolved fluorescence, as well as phosphorescence measurements, were used to resolve the luminescence properties of the three individual tryptophan residues of barnase. Assignment of the fluorescence properties was performed using single-tryptophan-containing mutants and the results were compared with the information available from the study of wild-type and two-tryptophan-containing mutants (Willaert, Lowenthal, Sancho, Froeyen, Fersht, Engelborghs, Biochemistry 1992;31:711-716). The fluorescence and the phosphorescence emission of wild-type barnase is dominated by Trp35, although Trp71 has the strongest intrinsic fluorescence when present alone. Fluorescence emission of these two tryptophan residues is blue-shifted and pH-independent. The fluorescence decay parameters of Trp94 are pH-dependent, and an intramolecular collision frequency of 2 to 5 x 10(9) s(-1) between Trp94 and His18 is calculated. Fluorescence emission of Trp94 is red-shifted. Fluorescence anisotropy decay reveals the local mobility of the individual tryptophan residues and this result correlates well with their phosphorescence properties. Trp35 and Trp71 display a single phosphorescence lifetime, which reflects the rigidity of their environment. Surface Trp94 does not exhibit detectable phosphorescence emission. The existence of energy transfer between Trp71 and Trp94, as previously detected by fluorescence measurements, is also observed in the phosphorescence emission of barnase. Dynamic quenching causes the phosphorescence intensity to be protein-concentration dependent. In addition, fluorescence anisotropy shows concentration dependency, and this can be described by the formation of trimers in solution.

Bacterial Proteins↗
PubMed1999

Tryptophan microstate reshuffling upon the binding of cyclosporin A to human cyclophilin A.

Human cyclophilin A (hCypA) contains one tryptophan residue at position 121 (Trp121). The fluorescence intensity of this single tryptophan residue doubles upon binding the clinically important immunosuppressant cyclosporin A (CsA). Trp121 is in close contact to the bound CsA and is well-conserved in almost all immunophilins. The enhancement of the fluorescence intensity upon binding CsA is investigated by steady-state and time-resolved fluorescence measurements. The crystal structures of hCypA and the complex hCypA-CsA are compared. Only Glu120 is strongly influenced by the binding of CsA. The distance between the indole ring and the carboxylate group doubles during complexation. The influence of Glu120 on the fluorescence properties of Trp121 was investigated by pH-titration, and by substituting glutamate into an aspartate and an alanine residue. The fluorescence measurements on the glutamate mutants reveal that the carboxylate group influences the fluorescence properties of Trp121 to a limited extent. The major effect of CsA binding, however, consists in a reshuffling of the populations of microconformations of Trp121 leading to a selective increase of the 1.5 ns lifetime component. This selection is also accompanied by a decreased polarity of the environment and an increase in the radiative rate constant.

Acrylamide↗
PubMed1999

Quantitative characterization of the binding of fluorescently labeled colchicine to tubulin in vitro using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a new technique that allows the determination of the diffusion constant of a fluorescent molecule in solution. Also, the binding of the fluorescent molecule to a target can be analyzed, if the difference in the diffusion coefficients of the free and bound ligand is sufficiently large. With FCS, the interaction between fluorescein-colchicine (FC) and tubulin has been studied in vitro. A fast and reversible binding is observed with an association constant at room temperature of (3.9 +/- 0.1) x 10(4) M-1. No competition with colchicine is seen, indicating that FCS reveals the existence of a new binding site on tubulin. FCS is not able to show the binding of FC to the original colchicine binding site, even though it exists, because the fluorescence of FC is strongly quenched upon binding to this site. This quenching is evident in spectrofluorometry experiments, revealing a slow binding of FC to tubulin that is subject to competition with colchicine. FCS allows the determination of the diffusion coefficients of both free and bound fluorescent colchicine which were found to be (2.6 +/- 0.2) x 10(-)10 and (2.0 +/- 0.2) x 10(-)11 m2 s-1, respectively. It can be concluded that fluorescent labeling, especially of small molecules, can interfere considerably with the binding behavior that is being studied. Although general qualitative effects in vivo are similar for colchicine and its fluorescein derivative, this quantitative study of the binding to tubulin presents a nuanced view, and the existence of a second binding site for FC can even explain some conflicting indications in the literature.

Animals↗
PubMed1999

Characterization of the hinges of the effector loop in the reaction pathway of the activation of ras-proteins. Kinetics of binding of beryllium trifluoride to V29G and I36G mutants of Ha-ras-p21.

This work experimentally confirms the pathway of activation of Ha-ras-p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434-451). The process can be studied experimentally by analyzing the binding of BeF3- to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138-23143). Two mutants, V29G and 136G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.

Beryllium↗
PubMed1998

General features of the recognition by tubulin of colchicine and related compounds.

The kinetic mechanisms of the binding to tubulin of colchicine and eight different analogues have been studied to elucidate details of the recognition mechanism. All of the analogues follow a two step binding mechanism i.e. binding occurs via an initial step with low affinity, followed by an isomerisation of the initial complex leading to the final high affinity state. For several analogues the kinetic and thermodynamic data of both processes are compared here. For all the analogues the delta G1 degree of initial binding at 25 degrees C varies between -13.3 and -28.8 kJ. mol-1. For the second step delta G2 degrees varies between -2.4 and -27 kJ. mol-1. These limited ranges of free energy change are, however, obtained by a great variety of enthalpy changes and compensatory entropy changes. Comparison of the data for the first and second steps indicates that structural alterations of the drugs always change the thermodynamic parameters of the two steps, and the changes in the first and the second steps are in opposite directions. The fact that this range of experimental behaviour can be incorporated into a general mechanism encourages the extension of these investigations to other colchicine analogues and related compounds with potential pharmaceutical applications.

Animals↗
PubMed1998

Comparative study of binase and barnase: experience in chimeric ribonucleases.

Chimeric enzymes were constructed to elucidate the differences in physicochemical properties of two related bacterial RNases, barnase and binase. Chimeras (Ba26Bi, Ba73Bi, Ba26Bi73Ba and Bi73Ba) contain six to thirteen residue substitutions relative to barnase, which are beyond the active site. The catalytic activity of RNases toward GpU, GpC and poly(I), as well as conformational distinctions and heat denaturation parameters, were studied. Thermal denaturation of binase, barnase and chimeric RNases is a two-state transition. The mutation-induced changes in the free energy of unfolding of barnase deduced from thermal and urea denaturation nearly coincide. The kinetic parameters for GpU and GpC demonstrate that the chimeras fall into two groups: barnase-like and binase-like. This division is determined by the origin of their C-terminal part (residues 73-110) which is also responsible for their thermostability at pH 2.4. An inverse linear dependence was found between kcat for poly(I) and denaturation temperature of RNases at pH 5.5, which points out that certain lability of the protein molecule appears to be necessary for efficient polynucleotide cleavage.

Amino Acid Sequence↗
PubMed1997

Mechanism of tubulin-colchicine recognition: a kinetic study of the binding of the colchicine analogues colchicide and isocolchicine.

Colchicide (IDE) is a colchicine (COL) analogue in which the C-10 methoxy group is replaced by a hydrogen atom. Its binding to tubulin is accompanied by a quenching of the protein fluorescence. The fluorescence decrease shows a monoexponential time dependence. The observed rate constant increases in a non-linear way with the total concentration of IDE, allowing the determination of a binding constant for an initial binding site (K1=5300+/-300 M-1) and the rate constant for the subsequent isomerization (k2=0.071+/-0.002 s-1) at 25 degrees C. The rate constant, k-2, for the reversed isomerization can be determined by displacement experiments. Despite the minor alteration of the C-ring substituent, the kinetic and thermodynamic parameters of binding are substantially different from those of COL itself, for both steps. In isocolchicine (ISO) the carbonyl oxygen atom and the methoxy groups of the C-ring have been interchanged. Its binding to tubulin only results in small fluorescence and absorbance changes. Therefore competition experiments with MTC [2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4, 6-cycloheptatrien-1-one] were performed. ISO competes rapidly and with low affinity with MTC. Fluorimetric titrations of tubulin with MDL (MDL 27048 or trans-1-(2,5 dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-propen-1 -one) in the presence and absence of ISO give evidence for the existence of a second, slow-reacting low-affinity site for ISO that is not accessible to MTC or MDL. The relevance of these results for the recognition of COL is analysed.

Animals↗
PubMed1997

The mechanism of tubulin-colchicine recognition--a kinetic study of the binding of a bicyclic colchicine analogue with a minor modification of the A ring.

2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-1-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25 degrees C were 540 +/- 65 M(-1) and 70 +/- 6 s(-1) respectively. From the temperature dependence, a reaction enthalpy change (deltaH(o)1) of the initial binding of 49 +/- 11 kJ/mol(-1) and an activation energy for the second step of 28 +/- 9 kJ/mol(-1) were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 +/- 0.07 s(-1) at 25 degrees C and the activation energy of 81 +/- 6 kJ/mol(-1). The overall binding constant was (6.3 +/- 0.2) x 10(4) M(-1) at 25 degrees C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A-ring colchicine analogues (podophyllotoxin, 3',4',5'-trimethoxyacetophenone and N-acetylmescaline) and a C-ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.

Animals↗
PubMed1997

Equilibrium and kinetic study of the conformational transition toward the active state of p21Ha-ras, induced by the binding of BeF3- to the GDP-bound state, in the absence of GTPase-activating proteins.

Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595-15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115-117). In this work, we have used a fluorescent active mutant (Y32W) of p21(Ha-)ras to demonstrate that BeF3- binds to the GDP. p21(Ha-ras) complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (Kd at 25 degrees C = 8.1 +/- 0.3 mM) and is endothermic (DeltaH = 22.3 +/- 1.6 kJ mol-1). The similarity between the GTP-bound form and the GDP.BeF3--bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP. BeF3-.p21(Ha-)ras complex to its final state. Both steps are endothermic (DeltaH1 = 15 +/- 2 kJ mol-1 and DeltaH2 = 8 +/- 2 kJ mol-1). The kinetically determined enthalpy change of 23 +/- 4 kJ mol-1 is in excellent agreement with the equilibrium analysis.

Anions↗
PubMed1997

Role of the Arg123-Tyr166 paired helix of apolipoprotein A-I in lecithin:cholesterol acyltransferase activation.

The Arg123-Tyr166 central and Ala190-Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) and apoA-I(Delta(Ala190-Gln243), nablaA-II(Ser12-Gln77)) chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight alpha-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 +/- 0.33 (mean +/- S.D.; n = 3), 0.054 +/- 0.009 (p < 0.001 versus apoA-I), and 1.3 +/- 0.32 (p = not significant versus apoA-I) nmol of cholesteryl ester/h/microM, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reduced Vmax with unaltered Km. Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) was very similar. In conclusion, although substitution of the Arg123-Tyr166 central or Ala190-Gln243 carboxyl-terminal pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I reduces the rate of LCAT activity that is independent of binding to rHDL.

Apolipoprotein A-I↗
PubMed1997

Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV).

Tubulin exists as various isoforms, which differ in their assembly, drug-binding properties, and the dynamic properties of the microtubules they compose. One of the most striking differences in drug binding among the isoforms is observed with colchicine, which binds much better to the alphabeta(II) and alphabeta(IV) isoforms than to the alphabeta(III) isoform. Here we have studied the interaction of these isoforms with 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone (MTPT), an analog of colchicine that lacks the B-ring. The kinetics of association and dissociation were studied fluorometrically, and the kinetic parameters for the two-step binding were determined for different beta-tubulin isoforms. The apparent on-rate constants for alphabeta(II), alphabeta(III) and alphabeta(IV) were 13358, 4558 and 10828 M(-1) s(-1), the off-rate constants (k(-2)) were 0.04, 0.03 and 0.02 s(-1), and the affinity constants are 3.33 x 10(5), 1.56 x 10(5) and 5.44 x 10(5) M(-1), respectively. The differences in kinetic parameters among different beta-tubulin isoforms are greatly reduced when the B-ring is removed. Our results indicate that the B-ring plays a major role in determining the isoform differences, and the results might be of importance for designing tissue-specific analogs of colchicine for cancer chemotherapy.

Colchicine↗
PubMed1997

Quenching of tryptophan fluorescence by the active-site disulfide bridge in the DsbA protein from Escherichia coli.

The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and required for efficient disulfide bond formation in the bacterial periplasm. The enzyme consists of a thioredoxin-like domain and a second, alpha-helical domain which is inserted into the thioredoxin motif. Reduction of the active-site disulfide in the thioredoxin domain causes a more than 3-fold increase in tryptophan fluorescence. However, both tryptophan residues of the protein, W76 and W126, are not in contact with the disulfide and located in the alpha-helical domain. Analysis of the variants W76F and W126F revealed that the fluorescence of W126 is fully quenched in every redox state of DsbA. W126 is also a sink for nonradiative energy transfer from W76. In oxidized DsbA, W76 is quenched by an intramolecular, dynamic quenching process which involves energy transfer from W76 via F26 to the disulfide. The contributions of the disulfide bridge and the tryptophan residues to the near-UV CD spectra were also quantified. Analysis of the thermodynamic stabilities of the variants W76F and F26L revealed that the interdomain contact between W76 and F26 strongly contributes to the overall stability of DsbA, and selectively stabilizes its oxidized form. The DsbA variant F26L is the most oxidizing disulfide oxidoreductase known so far.

Bacterial Proteins↗
PubMed1997

Response of microtubules to the addition of colchicine and tubulin-colchicine: evaluation of models for the interaction of drugs with microtubules.

The effects of free drug and tubulin-drug complexes on steady-state GTP/GDP-associated microtubules and on equilibrium guanosine 5'[beta,gamma-imido]triphosphate-associated microtubules are compared. The addition of colchicine or the tubulin-colchicine complex (TuCol) to steady-state microtubules induces microtubule disassembly. Only limited disassembly of equilibrium microtubules is observed under similar conditions. Addition of colchicine or the bifunctional colchicine analogue 2-methoxy-5-(2'3',4'-trimethoxyphenyl)tropone to preassembled steady-state or equilibrium microtubules does induce disassembly, but establishment of the new steady state or equilibrium is very slow. These observations are related to the fact that TuCol readily adds to the microtubule end, but is only incorporated into the lattice with difficulty. As a result, microtubule growth is effectively inhibited and the critical concentration is significantly increased. Nevertheless, drug-induced disassembly can be extremely slow, because the frequency of addition reactions increases as the concentration of soluble dimers increases. The efficiency of incorporation of TuCol decreases as it concentration increases. The work further confirms the existence of colchicine-binding sites with low affinity (association constant KMT approximately 3 x 10(2) M-1) along the microtubule lattice. This value suggests that part of the colchicine-binding site on tubulin remains available in the polymer. The interaction of colchicine with these sites has no appreciable effect on microtubule dynamics. These observations are reproduced and rationalized by the model described elsewhere [Vandecandelaere, Martin, Bayley and Schilstra (1994) Biochemistry 33, 2792-2801], and the possibility that there are co-operative effects in the inhibition is considered.

Animals↗
PubMed1997

Modelling pathways of alpha-chymotrypsin activation and deactivation.

The conformational change of alpha-chymotrypsin from an inactive, chymotrypsinogen like structure at high pH to an active conformation around pH 8.5 is used here as a model system to generate possible pathways for the transition by use of two different theoretical methods. One method, the 'targeted molecular dynamics' algorithm (TMD) adds a constraint in the direction of the target to a molecular dynamics force field and gives two different paths, one for every direction of the reaction (Schlitter,J., Engels,M., Krüger,P.J., Mol. Graphics (1994) 12, 84-89). The second method, the 'self penalty walk' algorithm (SPW), refines an initially guessed path by minimizing the sum of the energies of its structures (Elber,R. and Karplus,M., Chem. Phys. Lett. (1987) 139, 375-380). Thus, starting from a linear path as a first approximation, it produces a reaction coordinate of the transition. The structures of the TMD and SPW paths are similar only in the beginning while the middle part of the SPW path links the two TMD branches. The activation of alpha-chymotrypsin in the TMD path starts with a movement of loop VII (residues 215-225), pulling on loop VI (residues 186-194). Then the side chain of Met192 turns to the surface and Ile16 approaches Asp194 to form a salt bridge. In the TMD deactivation path, loop VII also moves and pushes loop VI to the protein core. The Met192 side chain adopts three intermediate conformations, till the salt bridge Ile16-Asp194 is broken and loop VI rearranges to its final conformation. In the SPW pathway both the formation of the salt bridge and the movement of Met192 happen simultaneously between two consecutive steps.

Algorithms↗
PubMed1997

Calculation of pathways for the conformational transition between the GTP- and GDP-bound states of the Ha-ras-p21 protein: calculations with explicit solvent simulations and comparison with calculations in vacuum.

The transitions between the water-equilibrated structures of the GTP and GDP forms of Ha-ras-p21 have been calculated by using the targeted molecular dynamics (TMD) method (Schlitter et al., Mol. Sim. 10:291-309, 1993) both in vacuo and with explicit solvent simulation. These constrained molecular dynamics calculations result in different pathways, depending on the nucleotide bound. Each pathway consists in a sequence of transitions affecting six segments of the protein, four of them forming a hydrophilic cleft around the nucleotide. The transitions are initiated by the removal or introduction of the gamma-phosphate of the nucleotide and proceed sequentially, crossing several low-energy transition states. The movements are transmitted either by direct interactions between the segments or through the nucleotide. The GTP to GDP pathway is initiated by the removal of the nucleotide gamma-phosphate. This gives some space to Gly12, Gly13, and Val14. Their movement is transmitted to the target recognition domain and the switch II region, forcing these segments to adopt another position. In a second step the target recognition domain and the switch II region undergo conformational transitions to reach an intermediate conformation. Finally, there is a relaxation of the target recognition domain to its final state that forces the switch II region to reach its target conformation. The calculated pathways allow the identification of many residues that play an important role in the conformational changes, explain the altered transformation properties of some, and suggest mutations to alter the pathway.

Cations, Divalent↗