Maturation efficiency, trypsin sensitivity, and optical properties of Arg96, Glu222, and Gly67 variants of green fluorescent protein

Spontaneous chromophore biosynthesis in green fluorescent protein (GFP) is initiated by a main-chain cyclization reaction catalyzed by the protein fold. To investigate the structural prerequisites for chromophore formation, we have substituted the conserved residues Arg96, Glu222, and Gly67. Upon purification, the variants can be ordered based on their decreasing extent of chromophore maturation according to the series EGFP, E222Q, R96K, G67A, and R96M. Arg96 and Glu222 appear to play catalytic roles, whereas Gly67 is likely important in interior packing to enforce correct hydrogen bonding to Arg96. The effect of Arg96 can be partially compensated for by a lysine, but not by a methionine residue, confirming its electrophilic role. Limited trypsinolysis data suggest that protein stability is largely unaffected by the presence of the chromophore, inconsistent with the mechanical compression hypothesis. Trends in optical properties may be related to the degree of chromophore charge delocalization, which is modulated by residue 96.     

Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis

Aequoria victoria green fluorescent protein (GFP) is a revolutionary molecular biology tool because of its spontaneous peptide backbone cyclization and chromophore formation from residues Ser65, Tyr66, and Gly67. Here we use structure-based design, comprehensive targeted mutagenesis, and high-resolution crystallography to probe the significant functional role of conserved Arg96 (R96) in chromophore maturation. The R96M GFP variant, in which the R96M side chain is similar in volume but lacks the R96 positive charge, exhibits dramatically slower chromophore maturation kinetics (from hours to months). Comparison of the precyclized conformation of the chromophore-forming residues with the mature R96M chromophore reveals a similar Y66 conformer, contrary to the large Y66 conformational change previously defined in the slowly maturing R96A variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. Comprehensive R96 mutagenesis and fluorescent colony screening indicate that only the R96K substitution restores wild-type maturation kinetics. Further, we show that the slowly maturing R96A variant can be complemented with a Q183R second-site mutation designed to restore the missing R96 positive charge and rapid fluorophore biosynthesis. Moreover, comparative structural analysis of R96M, R96K, R96A/Q183R, and wild-type GFP reveals the importance of the presence of positive charge, rather than its exact position. Together, these structural, mutational, and biochemical results establish a pivotal role for the R96 positive charge in accelerating the GFP post-translational modification, with implications for peptide backbone cyclization in GFP, its homologues, and related biological systems.     

Understanding the role of Arg96 in structure and stability of green fluorescent protein

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide-induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5- and 50-times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl-induced unfolding both in kinetics and in the quasi-equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP.     

The 2.1A crystal structure of copGFP, a representative member of the copepod clade within the green fluorescent protein superfamily

The green fluorescent protein (avGFP), its variants, and the closely related GFP-like proteins are characterized structurally by a cyclic tri-peptide chromophore located centrally within a conserved beta-can fold. Traditionally, these GFP family members have been isolated from the Cnidaria although recently, distantly related GFP-like proteins from the Bilateria, a sister group of the Cnidaria have been described, although no representative structure from this phylum has been reported to date. We have determined to 2.1A resolution the crystal structure of copGFP, a representative GFP-like protein from a copepod, a member of the Bilateria. The structure of copGFP revealed that, despite sharing only 19% sequence identity with GFP, the tri-peptide chromophore (Gly57-Tyr58-Gly59) of copGFP adopted a cis coplanar conformation within the conserved beta-can fold. However, the immediate environment surrounding the chromophore of copGFP was markedly atypical when compared to other members of the GFP-superfamily, with a large network of bulky residues observed to surround the chromophore. Arg87 and Glu222 (GFP numbering 96 and 222), the only two residues conserved between copGFP, GFP and GFP-like proteins are involved in autocatalytic genesis of the chromophore. Accordingly, the copGFP structure provides an alternative platform for the development of a new suite of fluorescent protein tools. Moreover, the structure suggests that the autocatalytic genesis of the chromophore is remarkably tolerant to a high degree of sequence and structural variation within the beta-can fold of the GFP superfamily.     

Guanidinium restores the chromophore but not rapid proton release in bacteriorhodopsin mutant R82Q.

Replacement of the Arg residue at position 82 in bacteriorhodopsin by Gln or Ala was previously shown to slow the rate of proton release and raise the pK of Asp 85, indicating that R82 is involved both in the proton release reaction and in stabilizing the purple form of the chromophore. We now find that guanidinium chloride lowers the pK of D85, as monitored by the shift of the 587-nm absorbance maximum to 570 nm (blue to purple transition) and increased yield of photointermediate M. The absorbance shift follows a simple binding curve, with an apparent dissociation constant of 20 mM. When membrane surface charge is taken into account, an intrinsic dissociation constant of 0.3 M fits the data over a range of 0.2-1.0 M cation concentration (Na+ plus guanidinium) and pH 5.4-6.7. A chloride counterion is not involved in the observed spectral changes, as chloride up to 0.2 M has little effect on the R82Q chromophore at pH 6, whereas guanidinium sulfate has a similar effect to guanidinium chloride. Furthermore, guanidinium does not affect the chromophore of the double mutant R82Q/D85N. Taken together, these observations suggest that guanidinium binds to a specific site near D85 and restores the purple chromophore. Surprisingly, guanidinium does not restore rapid proton release in the photocycle of R82Q. This result suggests either that guanidinium dissociates during the pump cycle or that it binds with a different hydrogen-bonding geometry than the Arg side chain of the wild type.     

A Molecular Mechanics and Database Analysis of the Structural Preorganization and Activation of the Chromophore-Containing Hexapeptide Fragment in Green Fluorescent Protein

Abstract

We propose that heterologous posttranslational chromophore formation in green fluorescent protein (GFP) occurs because the chromophore-forming amino acid residues 65SYG67 are preorganized and activated for imidazolinone ring formation. Based on extensive molecular mechanical conformational searching of the precursor hexapeptide fragment (64FSYGVQ69), we suggest that the presence of low energy conformations characterized by short contacts (～3Å) between the carbonyl carbon of Ser65 and the amide nitrogen of Gly67 accounts for the initial step in posttranslational chromophore formation. Database searches showed that the tight turn required to establish the key short contact is a unique structural motif that is rarely found, except in other FSYG tetrapeptide sequences. Additionally, ab initio calculations demonstrated that an arginine side chain can hydrogen bond to the carbonyl oxygen of Ser65, activating this group for nucleophilic attack by the nearby lone pair of the Gly67 amide nitrogen. We propose that GFP chromophore-formation is initiated by a unique combination of conformational and electronic enhancements, identified by computational methods.     

The crystal structure of the R52Q mutant demonstrates a role for R52 in chromophore pKa regulation in photoactive yellow protein

Mutating arginine 52 to glutamine (R52Q) in photoactive yellow protein (PYP) increases the pK(a) of the chromophore by 1 pH unit. The structure of the R52Q PYP mutant was determined by X-ray crystallography and was compared to the structure of wild-type PYP to assess the role of R52 in pK(a) regulation. The essential differences between R52Q and the wild type were confined to the loop region containing the 52nd residue. While the hydrogen bonds involving the chromophore were unchanged by the mutation, removing the guanidino group generated a cavity near the chromophore; this cavity is occupied by two water molecules. In the wild type, R52 forms hydrogen bonds with T50 and Y98; these hydrogen bonds are lost in R52Q. Q52 is linked to Y98 by hydrogen bonding through the two water molecules. R52 acts as a lid on the chromophore binding pocket and controls the accessibility of the exterior solvent and the pK(a) of the chromophore. R52 is found to flip out during the formation of PYP(M). The result of this movement is quite similar to the altered structure of R52Q. Thus, we propose that conformational changes at R52 are partly responsible for pK(a) regulation during the photocycle.     

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).     

Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry

The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.     

The role of conserved arginine residue in loop 4 of glycoside hydrolase family 10 xylanases

An arginine residue in loop 4 connecting beta strand 4 and alpha-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg(204) in xylanase A from Bacillus halodurans C-125 (XynA) and Arg(196) in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k(cat)/K(m) and the pH-k(cat) relationships of these mutant enzymes were measured. The pK(e2) and pK(es2) values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK(es2) value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK(a) value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.     

Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli

Site-directed mutagenesis was used to investigate the importance of Lys178, Arg286, and Arg294 in the binding of prephenate to the bifunctional enzyme chorismate mutase-prephenate dehydrogenase. From comparison of the kinetic parameters of wild-type enzyme and selected mutants, we conclude that only Arg294 interacts specifically with prephenate. The R294Q substitution reduces the enzyme's affinity for prephenate without affecting V/Et of the dehydrogenase reaction or the kinetic parameters of the mutase reaction. Arg294 likely interacts with the ring carboxylate at C-1 of prephenate since the dissociation constants for a series of inhibitors missing the ring carboxyl group were similar for wild-type and R294Q enzymes. The pH dependencies of log (V/KprephenateEt) and of pKi for hydroxyphenyllactate show that the wild-type dehydrogenase possesses a group with a pK of 8.8 that must be protonated for binding prephenate to the enzyme. None of the three conserved residues is this group since its titration is observed in the V/KprephenateEt profiles for the mutants K178Q, R286A, and R294Q. This group is also seen in the pH-rate profiles of the binding of two substrate analogues, hydroxyphenyllactate and deoxoprephenate. Their only common structural feature at C-1 is the side chain carboxylate, indicating that the protonated residue (pK 8.8) must interact with prephenate's side chain carboxylate. Gdn-HCl-induced denaturation was conducted on wild-type and selected mutant proteins. Unfolding of the wild-type enzyme proceeds through a partially unfolded dimer which dissociates into unfolded monomers. The order of stability is wild-type = R294Q > K178Q > R286A > K178R. The least unstable mutants have reduced mutase and dehydrogenase activities.     

The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in intrinsically disordered proteins with only local interactions

Detailed knowledge of the pH-dependence of ionizable residues in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein folding and stability. The reassembly of E. coli Thioredoxin (Trx) by complementation of its two disordered fragments (1-37/38-108) provides a folded heterodimer in equilibrium with its unfolded state which, based on circular dichroism and NMR spectroscopy, consists of two unfolded monomers. To gain insight into the role of electrostatics in protein folding and stability, we compared the pH-dependence of the carboxylate sidechain chemical shift of each Asp/Glu against that of its backbone amide chemical shift in the unfolded heterodimer. We monitored via C(CO)NH experiments four Asp and four Glu in fragments 38 to 108 (C37) of Trx in the pH range from 2.0 to 7.0 and compared them with results from (1)H(15)N HSQC experiments [Pujato et al., Biophys. J., 89 (2005) 3293-3302]. The (1)H(15)N HSQC analysis indicates two segments with quite distinct behavior: (A) a segment from Ala57 to Ala108 in which ionizable residues have up to three contiguous neighbors with pH-dependent backbone amide shifts, and (B) a segment of fifteen contiguous pH-dependent backbone amide shifts (Leu42 to Val56) in which two Asp and two Glu are implicated in medium range interactions. In all cases, the titration curves are simple modified sigmoidals from which a pH-midpoint (pH(m)) can be obtained by fitting. In segment A, the pH(m) of a given backbone amide of Asp/Glu mirrors within 0.15 pH-units that of its carboxylate sidechain (i.e., the pK(a)). In contrast, segment B shows significant differences with absolute values of 0.46 and 0.74 pH-units for Asp and Glu, respectively. The dispersion in the pH(m) of the backbone amide of Asp/Glu is also different in the two segments. Segment A shows a dispersion of 0.31 and 0.17 pH-units for Asp and Glu, respectively. Segment B shows a substantially larger dispersion (0.50 and 1.08 pH-units for Asp and Glu, respectively). In both segments, the dispersion in the pH(m) of its backbone amide is larger than in the pK(a) of the carboxylate sidechain (the latter is only 0.17 and 0.52 pH-units for Asp and Glu, respectively). Our results indicate that the pH(m) of the backbone amide chemical shift of Asp/Glu in a disordered polypeptide segment is a good predictor of its pK(a) whenever there are none or few neighboring backbone amides with similar pH-dependence.     

Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.     

Active site of epoxide hydrolases revisited: a noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate

The carboxylate of Glu35 in the active site of potato epoxide hydrolase StEH1 interacts with the catalytic water molecule and is the first link in a chain of hydrogen bonds connecting the active site with bulk solvent. To probe its importance to catalysis, the carboxylate was replaced with an amide through an E35Q mutation. Comparing enzyme activities using the two trans-stilbene oxide (TSO) enantiomers as substrates revealed the reaction with R,R-TSO to be the one more severely affected by the E35Q mutation, as judged by determined kinetic parameters describing the pre-steady states or the steady states of the catalyzed reactions. The hydrolysis of S,S-TSO afforded by the E35Q mutant was comparable with that of the wild-type enzyme, with only a minor decrease in activity, or a change in pH dependencies of kcat, and the rate of alkylenzyme hydrolysis, k3. The pH dependence of E35Q-catalyzed hydrolysis of R,R-TSO, however, exhibited an inverted titration curve as compared to that of the wild-type enzyme, with a minimal catalytic rate at pH values where the wild-type enzyme exhibited maximum rates. To simulate the pH dependence of the E35Q mutant, a shift in the acidity of the alkylenzyme had to be invoked. The proposed decrease in the pKa of His300 in the E35Q mutant was supported by computer simulations of the active site electrostatics. Hence, Glu35 participates in activation of the Asp nucleophile, presumably by facilitating channeling of protons out of the active site, and during the hydrolysis half-reaction by orienting the catalytic water for optimal hydrogen bonding, to fine-tune the acid-base characteristics of the general base His300.     

Glutamate 170 of human l-3-hydroxyacyl-CoA dehydrogenase is required for proper orientation of the catalytic histidine and structural integrity of the enzyme.

l-3-Hydroxyacyl-CoA dehydrogenase (HAD), the penultimate enzyme in the beta-oxidation spiral, reversibly catalyzes the conversion of l-3-hydroxyacyl-CoA to the corresponding 3-ketoacyl-CoA. Similar to other dehydrogenases, HAD contains a general acid/base, His(158), which is within hydrogen bond distance of a carboxylate, Glu(170). To investigate its function in this catalytic dyad, Glu(170) was replaced with glutamine (E170Q), and the mutant enzyme was characterized. Whereas substrate and cofactor binding were unaffected by the mutation, E170Q exhibited diminished catalytic activity. Protonation of the catalytic histidine did not restore wild-type activity, indicating that modulation of the pK(a) of His(158) is not the sole function of Glu(170). The pH profile of charge transfer complex formation, an independent indicator of active site integrity, was unaltered by the amino acid substitution, but the intensity of the charge transfer band was diminished. This observation, coupled with significantly reduced enzymatic stability of the E170Q mutant, implicates Glu(170) in maintenance of active site architecture. Examination of the crystal structure of E170Q in complex with NAD(+) and acetoacetyl-CoA (R = 21.9%, R(free) = 27.6%, 2.2 A) reveals that Gln(170) no longer hydrogen bonds to the side chain of His(158). Instead, the imidazole ring is nearly perpendicular to its placement in the comparable native complex and no longer positioned for efficient catalysis.     

Spectroscopic characterization of the photocycle intermediates of photoactive yellow protein.

The absorption spectra of photocycle intermediates of photoactive yellow protein mutants were compared with those of the corresponding intermediates of wild type to probe which amino acid residues interact with the chromophore in the intermediate states. B and H intermediates were produced by irradiation and trapped at 80 K, and L intermediates at 193 K. The absorption spectra of these intermediates produced from R52Q were identical to those from wild type, whereas those from E46Q and T50V were 7-15 nm red-shifted as those in the dark states. The absorption spectra of M intermediates were measured by flash photolysis at room temperature. Those of Y42F, T50V, and R52Q were identical to that of wild type, whereas that of E46Q was 11 nm red-shifted. Assuming that the intermediates of mutants have a structure comparable to that of wild type, these findings suggest the following: Glu46 interacts with the chromophore throughout the photocycle, interaction between the chromophore and Thr50 as well as Tyr42 is lost upon the formation of M intermediate, and Arg52 never interacts with the chromophore directly. The hydrogen-bonding network around the phenolic oxygen of the chromophore would be thus maintained until L intermediate decays, and the global conformational change would take place by the loss of the hydrogen bond between the chromophore and Tyr42. This model conflicts with some of the results of previous crystallographic studies, suggesting that the reaction mechanism in the crystal may be different from that in solution.     

pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates,chromophore deprotonation by hydroxyl uptake,and protonation relaxation of the dark state

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.     

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and spectral response of green fluorescent protein variants to changes in pH

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracellular pH. To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pattern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (S65G/V68L/S72A/T203Y) to change the solvent accessibility (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore. pH titrations of these variants indicate that the chromophore pKa can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9. Finally, mutagenesis was used to raise the pKa from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbestic point. This raises the interesting possibility of using fluorescence at this isosbestic point as an internal reference. Practical real time in vivo applications in cell and developmental biology are proposed.