The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.

Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.     

Tryptophan residues at subunit interfaces used as fluorescence probes to investigate homotropic and heterotropic regulation of aspartate transcarbamylase

The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-L-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.     

F1-ATPase changes its conformations upon phosphate release

Motor proteins, myosin, and kinesin have gamma-phosphate sensors in the switch II loop that play key roles in conformational changes that support motility. Here we report that a rotary motor, F1-ATPase, also changes its conformations upon phosphate release. The tryptophan mutation was introduced into Arg-333 in the beta subunit of F1-ATPase from thermophilic Bacillus PS3 as a probe of conformational changes. This residue interacts with the switch II loop (residues 308-315) of the beta subunit in a nucleotide-bound conformation. The addition of ATP to the mutant F1 subcomplex alpha3beta(R333W)3gamma caused transient increase and subsequent decay of the Trp fluorescence. The increase was caused by conformational changes on ATP binding. The rate of decay agreed well with that of phosphate release monitored by phosphate-binding protein assays. This is the first evidence that the beta subunit changes its conformation upon phosphate release, which may share a common mechanism of exerting motility with other motor proteins.     

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.     

Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop.     

Conformational state of the SecYEG-bound SecA probed by single tryptophan fluorescence spectroscopy

The SecYEG complex is a membrane-embedded channel that permits the passage of precursor proteins (preproteins) across the inner membrane of Escherichia coli. SecA is a molecular motor that associates with the SecYEG pore and drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the conformational state of soluble and SecYEG-bound SecA using single tryptophan mutants of SecA. The fluorescence spectral properties of the single tryptophans of SecA and their accessibility to the quencher acrylamide demonstrate that SecA undergoes a conformational change that results in a more compact structure upon binding of ATP and binding to the SecYEG pore. In addition, SecYEG-bound SecA undergoes ATP-dependent conformational changes that are not observed for soluble SecA. These data support a model in which binding to the SecYEG channel has a major impact on the SecA conformation.     

Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP. Ornithine and IMP function as activators, whereas UMP is an inhibitor. CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors. Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer. The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units. Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains. There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units. When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP. In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors. These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains. Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed. The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer, whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer. In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain. No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another.     

Steady-state kinetics and tryptophan fluorescence properties of halohydrin dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the active site and halide-induced conformational change

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 is a homotetrameric protein containing four tryptophan residues per subunit. The fluorescence properties of the enzyme are strongly influenced by halide binding. To examine the role of the tryptophans (W139, W192, W238, and W249) in halide binding and catalysis, they were individually mutated to a phenylalanine. All mutations, except for W238F, influenced the enzymatic properties. Mutating W192 to phenylalanine inactivated the enzyme and led to dissociation into dimers and monomers. In the structure of HheC, residue W139 and residue W249 from the opposite subunit are close to the active site of the enzyme. Substitution of W139 mainly affected K(m) values with all tested substrates and reduced the enantiopreference for p-nitro-2-bromo-1-phenylethanol. Replacing W249 increased both k(cat) and K(m) values with all tested substrates except for the (S)-enantiomer of p-nitro-2-bromo-1-phenylethanol, for which k(cat) was 3-fold decreased, resulting in a 6-fold increase of the enantioselectivity. Fluorescence measurements revealed that in the ligand-free state the intrinsic protein fluorescence of mutant W139F is higher than that of the wild-type enzyme, while the fluorescence intensity of mutants W238F and W249F was lower. The fluorescence intensities of the W238F and W249F enzymes were increased when they were unfolded or when bromide was added, whereas the fluorescence of mutant W139F was not increased by unfolding or addition of bromide. These results demonstrate that the fluorescence of residues W238 and W249 is partially quenched in the folded ligand-free state, and that W139 is completely quenched and acts as an energy acceptor for the other tryptophan residues as well. Changes of the maximum fluorescence emission wavelength of the HheC variants and the results of acrylamide quenching experiments confirmed that bromide binding induces a local conformational change around the active site, resulting in residue W139 and the quencher group being separated.     

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.     

Different classes of tryptophan residues involved in the conformational changes characteristic of the sarcoplasmic reticulum Ca2(+)-ATPase cycle

Various classes of tryptophan residues in the Ca2(+)-ATPase of sarcoplasmic reticulum membranes have been distinguished on the basis of their sensitivities to certain fluorescence quenchers: the brominated phospholipid 1,2-bis(9,10-dibromostearoyl)-sn-glycero(3)phosphocholine, the calcium ionophore calcimycin (A23187) and its brominated analog (4-bromo-A23187), and the nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate. We show that tryptophans located at the protein-lipid interface are the main contributors to the well-known fluorescence intensity change occurring in parallel with the conformational rearrangement induced by addition of calcium to the ATPase or its removal; Trp-794 on the ATPase chain may be one of these tryptophans. We also show that tryptophans more deeply embedded in the transmembrane protein structure contribute to the fluorescence change observed upon phosphorylation from inorganic phosphate of the calcium-free ATPase. This phosphorylation step involves opposite changes in the fluorescence quantum yield of tryptophans located in the membrane and in the cytoplasmic regions of the ATPase. This result is in agreement with models in which phosphorylation from inorganic phosphate not only changes the ATPase conformation locally around the catalytic center, but also reorganizes the membrane portion of the ATPase by long-range action, allowing, for instance, the calcium sites to become accessible from the luminal medium.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

Analysis of ATP binding to CheA containing tryptophan substitutions near the active site

Signal transduction in the chemotaxis system of Escherichia coli involves an autophosphorylating protein histidine kinase, CheA. At the active site of CheA, phenylalanine residues 455 and 459 occupy positions near the ATP-binding pocket, immediately adjacent to one of the hinge regions of a loop that undergoes an ATP-induced conformational change ("lid closure") that has been characterized previously in X-ray crystal structures [Bilwes et al. (2001) Nat. Struct. Biol. 8, 353-360]. We generated versions of CheA carrying F455W and F459W replacements and investigated whether the fluorescence properties of the introduced tryptophan side chains were affected by nucleotide binding in a manner that would provide a signal for investigating the dynamics of active site events, such as ATP binding and lid closure. Our results indicate that CheA(F455W) is useful in this regard, but CheA(F459W) is not. CheA(F455W) retained full catalytic activity and exhibited easily monitored fluorescence changes upon binding nucleotide: we observed a 25-30% decrease in CheA(F455W) fluorescence emission intensity at 330 nm upon binding ATP in the absence of Mg(2+); in the presence of Mg(2+), the emission spectrum of the CheA(F455W):ATP complex was red-shifted by 5 nm and exhibited an increased intensity (approximately 20% higher at 345 nm relative to that of uncomplexed CheA(F455W)). Different fluorescence changes were observed when two ATP analogues (ADPNP and ADPCP) were used instead of ATP and when Mn(2+) or Ca(2+) was used in place of Mg(2+). We exploited the fluorescence changes induced by Mg(2+)-ATP to explore the kinetics and mechanism of nucleotide binding by CheA(F455W). In the absence of Mg(2+), binding appears to involve a simple one-step equilibrium (k(assn) = 0.7 microM(-1) s(-1) and k(dissn) = 270 s(-1) at 4 degrees C). In the presence of Mg(2+), the binding mechanism involves at least two steps: (i) rapid, relatively weak binding followed by (ii) a rapid, reversible step (k(forward) = 300 s(-1) and k(reverse) =15 s(-1) at 4 degrees C) that enhanced the overall affinity of the complex and generated an increase in W455 fluorescence. This second step could reflect a conformational change at the CheA active site, such as lid closure. These results provide the first insight into the dynamics of nucleotide binding and substrate-induced conformational changes at the active site of a protein histidine kinase.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.     

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.     

Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.     

Ring up the curtain on DING proteins

We use site-directed mutagenesis to clarify the role of effector-mediated oligomerization changes on the modulation of the activity of Escherichia coli carbamoyl phosphate synthetase (CPS) by its allosteric activator ornithine and its inhibitor UMP. The regulatory domain mutations H975L, L990A and N992A abolished, and N987V decreased CPS oligomerization. The oligomerization domain mutation L421E prevented tetramer but not dimer formation. None of the mutations had drastic effects on enzyme activity or changed the sensitivity or apparent affinity of CPS for ornithine and UMP. Our findings exclude the involvement of oligomerization changes in the control of CPS activity, and show that CPS dimers are formed by the interactions across regulatory domains, and tetramers by the interactions of two dimers across the oligomerization domains. A mechanism for effector-mediated changes of the oligomerization state is proposed.     

Cofactor and DNA interactions in EcoRI DNA methyltransferase. Fluorescence spectroscopy and phenylalanine replacement for tryptophan 183.

EcoRI DNA methyltransferase contains tryptophans at positions 183 and 225. Tryptophan 225 is adjacent to residues previously implicated in S-adenosylmethionine (AdoMet) binding and to cysteine 223, previously shown to be the site of N-ethyl maleimide-mediated inactivation of the enzyme (Reich, N. O., and Everett, E. (1990) J. Biol. Chem. 265, 8929-8934; Everett, E. A., Falick, A. M., and Reich, N. O. (1990) J. Biol. Chem. 265, 17713-17719). The fluorescence spectra of the wild-type enzyme is centered at 338 nm indicating partial tryptophan solvent accessibility. Substitution of tryptophan 183 with phenylalanine results in a 45% drop in fluorescence intensity, but no shift in lambda max. DNA binding to the wild-type methyltransferase caused an increase in the fluorescence intensity, while binding to the tryptophan 183 mutant had a quenching effect, suggesting that DNA binding induces a conformational change near both tryptophans. Binding of AdoMet and various AdoMet analogs to the wild-type methyltransferase results in no change in the fluorescence spectrum when excitation occurs at 295 nm, suggesting that no conformational change occurs, and AdoMet does not interact with either tryptophan. In contrast, quenching was observed when excitation occurred at 280 nm, suggesting that AdoMet and its analogs may be quenching tyrosine to tryptophan energy transfer. Protein-ligand complexes were titrated with acrylamide, and the data also implicate conformational changes upon DNA binding but not upon AdoMet binding, consistent with previous limited proteolysis results (Reich, N. O., Maegley, K. A., Shoemaker, D.D., and Everett, E. (1991) Biochemistry 30, 2940-2946).     

Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes.

3-Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of 3-phosphoglycerate (3-PG) and ATP to 1,3-diphosphoglycerate (1,3-diPG) and ADP in the presence of magnesium ions. PGK is a single polypeptide chain arranged in two domains, with an active site located in the interdomain cleft. The large distance between the binding sites for 3-PG and ATP, deduced from the crystallographic structures of the binary complexes, gave rise to the hypothesis that this enzyme undergoes a hinge-bending domain motion from open to closed conformation during catalysis. However, no direct experimental evidence exists for the "closed" conformation in the presence of both substrates. In this study, several PGK mutants with single tryptophans placed in various location were used as intrinsic fluorescent probes to examine the extent and delocalization of conformational changes induced by the binding of 3-PG, 1,3-diPG, ADP, ATP, and PNP-AMP (nonhydrolyzable analogue of ATP), and by 3-PG and PNP-AMP together. The results showed that only the probes situated in the hinge and in parts of each domain close to the hinge reflect substrate-induced conformational changes. Binding of substrates to one domain was found to induce spectral perturbation of the probes in the opposite domain, indicating a transmission of conformational changes between the domains. A combination of both substrates generated much larger fluorescence changes than the individual substrates. The binding constants were determined for each substrate using probes situated in different locations.     

Tryptophan fluorescence of yeast actin resolved via conserved mutations

Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein. Replacement of each of these residues with either tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed the analysis of the contribution of these residues to the overall tryptophan fluorescence of actin. The sum of the relative contributions of these tryptophans was found to account for the intrinsic fluorescence of wild-type actin, indicating that energy transfer between the tryptophans is not the main determinant of their quantum yield, and that these mutations induce little conformational change to the protein. This was borne out by virtually identical polymerization rates and similar myosin interactions of each of the mutants and the wild-type actin. In addition, these mutants allowed the dissection of the microenvironment of each tryptophan as actin undergoes conformational changes upon metal cation exchange and polymerization. Based on the relative tryptophan contributions determined from single mutants, a triple mutant of yeast actin (W79) was generated that showed small intrinsic fluorescence and should be useful for studies of actin interactions with actin-binding proteins.