The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine

A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.     

Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.     

Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants.

Time correlated single photon counting measurements of tryptophan (Trp) fluorescence intensity decay and other spectroscopic studies were performed on glutamine-binding protein (GlnBP) from Escherichia coli. Using site-specifically mutated forms of the protein in which tyrosine (Tyr) and phenylalanine (Phe) substitute for the Trp residues at positions 32 and 220, we have examined whether wild-type (Wtyp) intensity decay components may be assigned to specific Trp residues. Results indicate that: (a) two exponential intensity decay components are recovered from the Wtyp protein (6.16 ns, 0.46 ns); (b) the long decay component arises from Trp-220 and comprises greater than 90% of the total fluorescence emission; (c) the short component arises from Trp-32 and is highly quenched; (d) all four single-Trp mutants exhibit multiexponential intensity decays, yet equimolar mixtures of two single-Trp mutants yield only two decay components which are virtually indistinguishable from the Wtyp protein; (e) the recovery of additional components in protein mixtures is obscured by statistical noise inherent in the technique of photon counting; (f) various spectroscopic measurements suggest that Trp-Trp interactions occur in the Wtyp protein, but the Wtyp intensity decay may be closely approximated by a linear combination of intensity decays from single-Trp mutants; and (g) inferences derived independently from fluorescence and NMR spectroscopy which pertain to the presence of Trp-Trp interactions and the relative solvent exposure of the two Trp residues are in agreement.     

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.     

Intrinsic fluorescence of globular actin. Features of localization of tryptophan residues

The contribution of individual Trp residues to alpha-actin fluorescence was evaluated by means of an analysis of their microenvironment, which was done on the basis of PIR-International protein sequence database information. The contribution of Trp79 and Trp86 was shown to be low due to an effective nonradiating energy transfer according to the inductive resonance mechanism between the Trp residues and the fluorescence quenching of Trp86 by S gamma of Cys10, an efficient fluorescence quencher. The intrinsic fluorescence of actin was found to be determined mainly by Trp340 and Trp356, which are internal, inaccessible to solvent, and have a high density microenvironment formed mainly by nonpolar groups of protein. It is possible that the side chain conformation of Trp340 (t-isomer; chi 1 190 degrees, chi 2 89 degrees), aromatic rings of Tyr and Phe residues, and Pro residues in the microenvironment of Trp340 and Trp356 substantially contribute to the short-wavelength fluorescence spectrum of actin.     

Fluorescence spectra of luteinizing hormone releasing hormone,a decapeptide containing histidyl,tryptophyl and tyrosyl residues: Analysis by derivative spectroscopy

Fluorescence spectra have been obtained for luteinizing hormone releasing hormone, a decapeptide containing His, Trp and Tyr, and analogs lacking one or more of these residues. The second derivatives of these spectra were used to examine the contributions of the three residues to the spectrum of the hormone. Tyr influences the excitation spectrum when fluorescence is monitored at an emission wavelength of 305 nm but makes little or no contribution to the emission spectrum when the compound is excited at 275 nm. His and Trp influence both excitation and emission spectra.     

Role of hydrogen bonding in the active site of human manganese superoxide dismutase

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.     

Functional and structural consequences of aromatic residue substitutions within the kringle-2 domain of tissue-type plasminogen activator.

Aromatic amino acid residues within kringle domains play important roles in the structural stability and ligand-binding properties of these protein modules. In previous investigations, it has been demonstrated that the rigidly conserved Trp25 is primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2tpA), whereas Trp63, Trp74, and Tyr76 function in omega-amino acid ligand binding, and, to varying extents, in stabilizing the native folding of this kringle module. In the current study, the remaining aromatic residues of K2tPA, viz., Tyr2, Phe3, Tyr9, Tyr35, Tyr52, have been subjected to structure-function analysis via site-directed mutagenesis studies. Ligand binding was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr35 destabilized the interaction of the ligand with the variant kringle. In addition, as reflected in the values of the melting temperatures, changes at Tyr9 and Tyr52 generally destabilized the native structure of K2tPA to a greater extent than changes at Tyr2, Phe3, and Tyr35. Taken together, results to date show that, in concert with predictions from the crystal structure of K2tpA, ligand binding appears to rely most on the integrity of Trp63 and Trp74, and aromaticity at Tyr76. With regard to aromatic amino acids, kringle folding is most dependent on Tyr9, Trp25, Tyr52, Trp63, and Tyr76. As yet, no obvious major roles have been uncovered for Tyr2, Phe3, or Tyr35 in K2tpA.     

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.     

Controlling ligand binding in myoglobin by mutagenesis.

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.     

Spectral contribution of the individual tryptophan of alphaB-crystallin: a study by site-directed mutagenesis

There are two tryptophan residues in the lens alphaB-crystallin, Trp9 and Trp60. We prepared two Trp --> Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near-ultraviolet circular dichroism (UV CD) differently. The major difference in the near-UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near-UV CD shows an increased intensity in the region of 270-280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a beta-strand.     

Critical amino acids for the 8(R)-dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases

7,8-Linoleate diol synthase (7,8-LDS) of the take-all fungus and cyclooxygenases can be aligned with approximately 24% amino acid identity and both form a tyrosyl radical during catalysis. 7,8-LDS was expressed in insect cells with native 8R-dioxygenase and hydroperoxide isomerase activities. We studied conserved residues of 7,8-LDS, which participate in cyclooxygenases for heme binding (His residues), hydrogen abstraction (Tyr), positioning (Tyr, Trp), and ionic binding of substrates (Arg). Site-directed mutagenesis abolished 8R-dioxygenase activities with exception of the putative distal histidine (His203Gln) and a tyrosine residue important for hydrogen bonding and substrate positioning (Tyr329Phe). The results demonstrate structural similarities between 7,8-LDS and cyclooxygenases.     

The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-A resolution

An atomic model of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been built into an electron density map at 2.4 A resolution, using chemical sequences of Mn dismutases from Thermus aquaticus and Bacillus stearothermophilus. The monomer fold is structurally very similar to the fold of iron dismutase and comprises two domains, each contributing two ligands to the metal. The Mn(III) ion is bound by protein ligands assigned as His 28, His 83, Asp 165, and His 169. Near neighbors in the metal-ligand environment include a series of hydrophobic residues, Phe 86, Trp 87, Trp 131, and Trp 167. The hydroxyl groups of two Tyr residues, at 36 and 182, are less than 7 A from the metal, as is His 32. Gln 150 forms a bridge between Tyr 36 and Trp 131. These ligands and nearby residues are strongly conserved in the known sequences of Mn dismutases. Only one of the two oxygens of Asp 165 has been assigned as a metal ligand, so that in the current model four protein atoms bind Mn(III). These ligand atoms form part of an approximate trigonal bipyramid in which water may occupy an axial position on the side opposite His 28. The conformation of the protein is unusual in the vicinity of the first ligand, His 28, as a consequence of the insertion of an extra residue in an alpha-helix. The distortion of the helix allows His 32 to stack against the ligand, His 169, and brings Tyr 36 close to the Mn ion. Across one of the dimer interfaces, the two Mn ions are separated by about 18 A, and active center residues from adjoining subunits interdigitate; Tyr 172 interacts with His 32 of the neighboring chain and Glu 168 with the backbone of 168 and with the ligand His 169 from the opposite subunit. Only one other dimer interface occurs in the tetramer; it involves residues 55-62 and sequences near 140 and 156. The center of the oligomeric molecule is filled with solvent.     

Computational studies of pandemic 1918 and 2009 H1N1 hemagglutinins bound to avian and human receptor analogs

The purpose of this work was to study the binding properties of two pandemic influenza A virus 1918 H1N1 (SC1918) and 2009 H1N1 (CA09) hemagglutinin (HA) with avian and human receptors. The quantum chemical calculations have been performed to analyze the interactions of 130 loop, 190 helix, 220 loop region, and conserved residues 95,145,153–155, of pandemic viruses’ HA with sialo-trisaccharide receptor of avian and human using density functional theory. The HA’s residues Tyr 95, Ala 138, Gln 191, Arg 220, and Asp 225 from the above regions have stronger interaction with avian receptor. The residues Thr 136, Trp 153, His 183, and Asp 190 of HA are important and play a significant role to bind with human receptor. The residues Tyr 95, Ala 138, Lys 145, Trp 153, Gln 192, and Gln 226 of HA of CA09 virus have found more interaction energies with human than avian receptors. Due to mutations in the active residues of HA of CA09 virus comparing with SC1918, the binding capabilities of HA with human have been increased. The molecular dynamics simulation was made to understand the different dynamical properties of HA and molecular interactions between HA of these two viruses with sialo-trisaccharide receptors of avian and human receptors. The interaction energy of HA of CA09 virus with human receptor decreases due to the human receptor far away from conserved residue region of HA protein. This reveals that the conserved residues particularly Lys 145 play major contribution to interaction with human receptor in HA of CA09 virus.     

Fluorescence and excitation Escherichia coli RecA protein spectra analyzed separately for tyrosine and tryptophan residues

The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described. The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg(2+), ATPgammaS or ADP, and single-stranded DNA (ssDNA). RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R. M. Story, I. T. Weber, and T. A. Steitz (1992) Nature (London) 355, 374-376). Because the fluorescence of Tyr residues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively. Spectral analysis of Trp and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATPgammaS or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA. Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA. The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATPgammaS or ADP. By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine. In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectra were substituted by the spectral function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain.     

Fluorescence contributions of the individual Trp residues in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.     

Tryptophan mutants of intestinal fatty acid-binding protein: ultraviolet absorption and circular dichroism studies.

An UV absorption and CD study of intestinal fatty acid-binding protein is presented. Since there are only two Trp residues in the molecule, two single-Trp mutants were prepared to deconvolute their signals. The individual contribution of the eight Phe and four Tyr residues was not established; however, Phe global contribution is relatively free of interferences from the other chromophores and was observed directly. CD spectra showed that Phe vibronic structure was unusually sharp and seems to monitor very specific details in the three-dimensional structure. The global signal from Tyr was assigned only approximately due to band broadening and overlapping. At the upper end of the CD spectrum, strong positive (1)L(b) Trp transitions from Trp 82 and strong negative (1)L(b) Trp transitions from Trp 6 were observed. (1)L(a) transitions were overall weak, positive for Trp 82 and negative for Trp 6, nearly cancelling each other out in the final spectrum. The above assignment is of practical and fundamental interest to monitor folding, binding, and molecular dynamics down to microdomain resolution. The assignment of Trp bands allowed comparison with previous data from CRABP1, another member of the IFABP family with 28% identical residues. It was found that structural homology extends beyond sequence and tertiary fold to include optical properties of equivalent Trp residues in the structure.     

Investigations on C-H...pi interactions in RNA binding proteins

We have investigated the roles played by C-Hcdots, three dots, centeredpi interactions in RNA binding proteins. There was an average of 55 C-Hcdots, three dots, centeredpi interactions per protein and also there was an average of one significant C-Hcdots, three dots, centeredpi interaction for every nine residues in the 59 RNA binding proteins studied. Main-chain to side-chain C-Hcdots, three dots, centeredpi interactions is the predominant type of interactions in RNA binding proteins. The donor atom contribution to C-Hcdots, three dots, centeredpi interactions was mainly from Phe, Tyr, Trp, Pro, Gly, Lys, His and Ala residues. The acceptor atom contribution to main-chain to side-chain C-Hcdots, three dots, centeredpi and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions was mainly from Phe and Tyr residues. On the contrary, the acceptor atoms of Trp residues contributed to all the four types of C-Hcdots, three dots, centeredpi interactions. Also, Trp contributed both donor and acceptor atoms in main-chain to side-chain, main-chain to side-chain five-member aromatic ring and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions. The secondary structure preference analysis of C-Hcdots, three dots, centeredpi interacting residues showed that, Arg, Gln, Glu, His, Ile, Leu, Lys, Met, Phe and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Trp and Val preferred to be in strand conformation. Long-range C-Hcdots, three dots, centeredpi interactions are the predominant type of interactions in RNA binding proteins. More than 50% of C-Hcdots, three dots, centeredpi interacting residues had a higher conservation score. Significant percentage of C-Hcdots, three dots, centeredpi interacting residues had one or more stabilization centers. Seven percent of the theoretically predicted stabilizing residues were also involved in C-Hcdots, three dots, centeredpi interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Interaction of firefly luciferase with substrates and their analogs: a study using fluorescence spectroscopy methods.

The results of the author's laboratory on the interaction of Luciola mingrelica firefly luciferase with substrates and their analogs using both steady-state and time resolved fluorescence are reviewed. The contribution of fluorescence of Trp and Tyr residues of the protein to its intrinsic fluorescence spectrum was estimated. Studies of quenching of Trp and Tyr fluorescence by luciferin and ATP allowed one to determine binding constants of the luciferase with substrates and to show that the binding of one substrate to the luciferase decreases the affinity of the enzyme for the other one. Fluorescence of oxyluciferin and its analogs (dimethyl- and monomethyloxyluciferins) was shown to be a good model of native firefly bioluminescence. A comparison of the fluorescence spectra of oxyluciferin and its analogs in aqueous solutions and in the presence of the luciferase revealed specific and nonspecific effects of the microenvironment on the equilibrium between different ionic forms of oxyluciferin. An approach based on photo-physical concepts of the correlation between luminescence spectra and structure of the emitter and its microenvironment was proposed and this approach was used to analyze bioluminescence spectra of wild-type and mutant luciferases.     

Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability, and folding has been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered 17 conserved sequence positions and three nonconserved positions. As a first step, the influence of the mutations on the protein folding reaction has been probed, revealing a folding nucleus of eight hydrophobic residues formed between the N- and C-terminal helices [Kragelund, B. B., et al. (1999) Nat. Struct. Biol. (In press)]. To fully analyze the role of the conserved residues, the function and the stability have been measured for the same set of mutant proteins. Effects on function were measured by the extent of binding of the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. The sequence sites that have been conserved for direct functional purposes have been identified. These are Phe5, Tyr28, Tyr31, Lys32, Lys54, and Tyr73. Binding site residues are mainly polar or charged residues, and together, four of these contribute approximately 8 kcal mol-1 of the total free energy of binding of 11 kcal mol-1. The sequence sites conserved for stability of the structure have likewise been identified and are Phe5, Ala9, Val12, Leu15, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially, all of the conserved residues that maintain the stability are hydrophobic residues at the interface of the helices. Only one conserved polar residue, Gln33, is involved in stability. The results indicate that conservation of residues in homologous proteins may result from a summed optimization of an effective folding reaction, a stable native protein, and a fully active binding site. This is important in protein design strategies, where optimization of one of these parameters, typically function or stability, may influence any of the others markedly.