Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis.

The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells. Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase.     

Probing local environments of tryptophan residues in proteins: comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor

19F nuclear magnetic resonance (19F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trpl4 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J 1995;69:20-29). 19F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain.     

Neurospora Mutant Deficient in Tryptophanyl-Transfer Ribonucleic Acid Synthetase Activity

A tryptophan auxotroph of Neurospora crassa, trp-5, has been characterized as a mutant with a deficient tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.2) activity. When assayed by tryptophanyl-tRNA formation, extracts of the mutant have less than 5% of the wild-type specific activity. The adenosine triphosphate-pyrophosphate exchange activity is at about half the normal level. In the mutant derepressed levels of anthranilate synthetase and tryptophan synthetase were associated with free tryptophan pools equal to or higher than those found in the wild type. We conclude that a product of the normal tryptophanyl-tRNA synthetase, probably tryptophanyl-tRNA, rather than free tryptophan, participates in the repression of the tryptophan biosynthetic enzymes.     

Identity elements and aminoacylation of plant tRNATrp.

Mutation of the Arabidopsis thaliana tRNA (Trp)(CCA) anticodon or of the A73 discriminator base greatly diminishes in vitro aminoacylation with tryptophan, indicating the importance of these nucleotides for recognition by the plant tryptophanyl-tRNA synthetase. Mutation of the tRNA (Trp)(CCA) anticodon to CUA so as to translate amber nonsense codons permits tRNA (Trp)(CCA) to be aminoacylated by A.thaliana lysyl-tRNA synthetase. Thus, translational suppression by tRNA (TRP)(CCA) observed in plant cells includes significant incorporation of lysine into protein.     

Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended beta1-beta2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA(Trp), which could be partially rescued by adding tRNA(Trp), even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.     

Fluorescence based structural analysis of tryptophan analogue-AMP formation in single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase

The symmetrical dimer structure of tryptophanyl-tRNA synthetase is similar to that of tyrosyl-tRNA synthetase whose binding behavior and structural details have been elucidated in detail. The structure of both subunits after forming the intermediate tryptophanyl-AMP has important implications for the binding of the cognate tRNA(Trp). Single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase have been constructed and expressed and used to probe structural changes in different domains of the enzyme in both subunits. Substrate titrations using the Trp analogues 4-fluorotryptophan and 7-azatryptophan in the presence of ATP to form the corresponding aminoacyl-adenylate reveal significant structural changes occurring throughout the active subunit in regions not confined to the active site. Changes in environment around the specific Trp residues were monitored using UV absorbance and steady-state fluorescence measurements. When titrated with 4-fluorotryptophan, both Trp 91 and Trp 290 fluorescence is quenched (49 and 22%, respectively) when one subunit has formed Trp-AMP. The fluorescence of Trp 48 is enhanced 19%. No further change in signal was observed after a 1:1 dimer/L-4FW-AMP complex ratio had been established. Using an anion-exchange filter binding assay with radiolabeled l-Trp as a substrate, binding to only one subunit was observed under nonsaturating conditions. This agrees with the results of the assay using 7-azatryptophan as a substrate. The observed changes extend to the unfilled subunit where a similar structure is believed to form after one subunit has formed tryptophan-AMP. Movement in the regions of the enzyme containing Trp 290 and Trp 91 suggests a mechanism for cross-subunit communication involving the helical backbone and dimer interface containing these two residues.     

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic model describing the action of tryptophanyl-tRNA-synthetase]

Reaction rates for ATP-PPi isotope exchange (vex) and tryptophanyl-tRNA formation (vaa) catalysed concomitantly in one incubation mixture by beef pancreas tryptophanyl-tRNA synthetase (trsase) have been examined as a function of substrate concentrations. Comparison of the vex/vaa ratio found experimentally with the ratio predicted theoretically conforms the mechanism suggested earlier and permits to describe it in more detail. I. At least two reaction routes exist in which an ATP-PP: exchange is allowed. These routes are interconnected with each other via the stage at which tRNA binds to the enzyme. 2. In both these routes the low molecular weight substrates bind with enzyme in the order ATP first, tryptophan second. 3. Enzyme-aminoacyladenylate complex is an intermediate in the reaction of aminoacyl-tRNA formation. Pyrophosphate is detached from the enzyme prior to tRNA. 4. The enzyme releases AMP and tryptophanyl-tRNA in a random fashion. All the aformentioned properties are common both for trigger mechanism and Yarus-Berg mechanism which up to now were considered in literature independently.     

Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue

Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.     

A Concerted Tryptophanyl-adenylate-dependent Conformational Change inBacillus subtilisTryptophanyl-tRNA Synthetase Revealed by the Fluorescence of Trp92

A semi-conserved tryptophan residue ofBacillus subtilistryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNA^Trpbinding and recognition. The crystal structure of theBacillus stearothermophilusTrpRS tryptophanyl-5′-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation. Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy.B. subtilisTrpRS has a single tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5′-adenylate (4FW-AMP) TrpRS complex. Complexation causes the Trp92 fluorescence to become quenched by 70%. Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled. This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes. Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form. From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNA^Trpis unlikely.Time-resolved fluorescence indicates conformational heterogeneity ofB. subtilisTrp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed- TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure.The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an α-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 Å) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn apart. If this local α-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues. Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92. A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small α-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit. This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding. We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.     

Regioselective nitration of tryptophan by a complex between bacterial nitric-oxide synthase and tryptophanyl-tRNA synthetase

Bacterial nitric-oxide synthase proteins (NOSs) from certain Streptomyces strains have been shown to participate in biosynthetic nitration of tryptophanyl moieties in vivo (Kers, J. A., Wach, M. J., Krasnoff, S. B., Cameron, K. D., Widom, J., Bukhaid, R. A., Gibson, D. M., and Crane, B. R., and Loria, R. (2004) Nature 429, 79-82). We report that the complex between Deinococcus radiodurans NOS (deiNOS) and an unusual tryptophanyl-tRNA synthetase (TrpRS II) catalyzes the regioselective nitration of tryptophan (Trp) at the 4-position. Unlike non-enzymatic Trp nitration, and similar reactions catalyzed by globins and peroxidases, deiNOS only produces the otherwise unfavorable 4-nitro-Trp isomer. Although deiNOS alone will catalyze 4-nitro-Trp production, yields are significantly enhanced by TrpRS II and ATP. 4-Nitro-Trp formation exhibits saturation behavior with Trp (but not tyrosine) and is completely inhibited by the addition of the mammalian NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B). Trp stimulates deiNOS oxidation of substrate l-arginine (Arg) to the same degree as H(4)B. These observations are consistent with a mechanism where Trp or a derivative thereof binds in the NOS pterin site, participates in Arg oxidation, and becomes nitrated at the 4-position.     

Catalytic mechanism of the tryptophan activation reaction revealed by crystal structures of human tryptophanyl-tRNA synthetase in different enzymatic states

Human tryptophanyl-tRNA synthetase (hTrpRS) differs from its bacterial counterpart at several key positions of the catalytic active site and has an extra N-terminal domain, implying possibly a different catalytic mechanism. We report here the crystal structures of hTrpRS in complexes with Trp, tryptophanamide and ATP and tryptophanyl-AMP, respectively, which represent three different enzymatic states of the Trp activation reaction. Analyses of these structures reveal the molecular basis of the mechanisms of the substrate recognition and the activation reaction. The dimeric hTrpRS is structurally and functionally asymmetric with half-of-the-sites reactivity. Recognition of Trp is by an induced-fit mechanism involving conformational change of the AIDQ motif that creates a perfect pocket for the binding and activation of Trp and causes coupled movements of the N-terminal and C-terminal domains. The KMSAS loop appears to have an inherent flexibility and the binding of ATP stabilizes it in a closed conformation that secures the position of ATP for catalysis. Our structural data indicate that the catalytic mechanism of the Trp activation reaction by hTrpRS involves more moderate conformational changes of the structural elements at the active site to recognize and bind the substrates, which is more complex and fine-tuned than that of bacterial TrpRS.     

Temperature-induced derepression of tryptophan biosynthesis in a tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (l-tryptophan: tRNA ligase adenosine monophosphate, EC 6.1.1.2) mutant (trpS1) of Bacillus subtilis is derepressed for enzymes of the tryptophan biosynthetic pathway at temperatures which reduce the growth rate but still allow exponential growth. Derepression of anthranilate synthase in a tryptophan-supplemented medium (50 mug/ml) is maximal at 36 C, and the differential rate of synthesis is 600- to 2,000-fold greater than that of the wild-type strain or trpS1 revertants. A study of the derepression pattern in the mutant and its revertants indicates that the 5-fluorotryptophan recognition site of the tryptophanyl-tRNA synthetase is an integral part of the repression mechanism. Evidence for a second locus, unlinked to the trpS1 locus, which functions in the repression of tryptophan biosynthetic enzymes is presented.     

Docking of tryptophanyl [corrected tryptophan] analogs to trytophanyl-tRNA synthetase: implications for non-canonical amino acid incorporations

Abstract Non-canonical amino acids (N(AA)), as building blocks for peptides and proteins during ribosomal translation, represent a nearly infinite supply of novel functions. The specific selection, activation and tRNA-charging of amino acids by aminoacyl-tRNA synthetases (AARS) in the aminoacylation reaction are essential steps. In most cases, aminoacylation of N(AA) is a good indication that the related amino acid will participate in ribosomal translation as well. However, testing the translational capacity of amino acid analogs has technical limitations. Therefore, a rapid and reliable in silico test for N(AA) recognition by AARS would be advantageous in experimental design. We chose tryptophanyl-tRNA synthetase from Escherichia coli as a model system for docking studies with various tryptophan analogs using the FlexX-Pharm strategy. We were able to calculate relative binding energies for Trp analogs in TrpRS that correlate well with their translational activities in E. coli. In particular, FlexX-Pharm predicted the binding sites of fluoro-, amino-, hydroxyl- and aza-containing Trp analogs within 1.5 A of Trp in the homology model of E. coli TrpRS. Therefore, the use of ligand docking prior to N(AA) incorporation experiments might provide a straightforward means for determining N(AA) that can be efficiently incorporated into a protein.     

Selection of cell lines resistant to tryptophan analogs

After prolonged cultivation in the presence of increasing amounts of carboxyl-substituted tryptophan analogs (tryptamine and tryptophanol), cell lines resistant to high concentrations of these compounds were obtained. The initial culture was the Madin-Darby line of spontaneously transformed bovine kidney cells. In the resistant lines the amount of tryptophanyl-tRNA synthetase (E. C. 6.1.1.2) is manyfold increased as shown by two criteria: (i) enzymatic activity (ATP-PPi isotopic exchange) per mg of protein, (ii) binding of in vivo 35S-labeled proteins to polyclonal antibodies against tryptophanyl-tRNA synthetase. It was shown that tryptophanyl-tRNA synthetase is phosphorylated in vivo, and the degree of phosphorylation of the enzyme in initial cells seems to be higher then in the resistant ones. The Km value for tryptophan is not significantly changed for the enzyme from resistant cells. The permeability for tryptophan and its analogs is reduced in the resistant cells. It is proposed that the acquisition of the resistance against tryptophan analogs are due to alterations at the genomic level (for example, gene amplification etc.).     

Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas

The formation of tryptophanyl adenylate catalyzed by tryptophanyl-tRNA synthetase from beef pancreas has been studied by stopped-flow analysis under conditions where the concentration of one of the substrates was largely decreasing during the time course of the reaction. Under such conditions a nonlinear regression analysis of the formation of the adenylate (adenylate vs. time curve) at several initial tryptophan and enzyme concentrations gave an accurate determination of both binding constants of this substrate. The use of the jackknife procedure according to Cornish - Bowden & Wong [ Cornish - Bowden , A., & Wong , J.J. (1978) Biochem. J. 175, 969-976] gave the limit of confidence of these constants. This approach confirmed that tryptophanyl-tRNA synthetase presents a kinetic anticooperativity toward tryptophan in the activation reaction that closely parallels the anticooperativity found for tryptophan binding at equilibrium. Both sites are simultaneously forming the adenylate. The dissociation constants obtained under the present pre-steady-state conditions for tryptophan are KT1 = 1.6 +/- 0.5 microM and KT2 = 18.5 +/- 3.0 microM at pH 8.0, 25 degrees C. The rate constant kf of adenylate formation is identical for both active sites (kf = 42 +/- 5 s-1). The substrate depletion method presently used, linked to the jackknife procedure, proves to be particularly suitable for the determination of the kinetic constants and for the discrimination between different possible kinetic models of dimeric enzyme with high substrate affinity. In such a case this method is more reliable than the conventional method using substrate concentrations in high excess over that of the enzyme.     

19F NMR studies of plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is a 43 kDa protein involved in the regulation of fibrinolysis. PAI-1 is the principal inhibitor of tissue-type plasminogen activator (t-PA), trapping the proteinase as an acyl-enzyme covalent complex (approximately 105 kDa). Four single tryptophan mutants of PAI-1 have been constructed in which three of the four tryptophan residues (Trp86, Trp139, Trp175, and Trp262) were replaced with phenylalanine. Biosynthetic incorporation of 5-fluorotryptophan (5F-Trp) into wild-type PAI-1 (5FW wtPAI-1) and the single tryptophan mutants (5FW86, 5FW139, 5FW175, and 5FW262) was achieved, allowing a (19)F NMR spectroscopic study of PAI-1 in its active and cleaved forms and in complex with t-PA. The (19)F NMR spectrum of active 5FW wtPAI-1 shows four clearly resolved peaks at -39.20, -49.26, -50.74, and -52.57 ppm relative to trifluoroacetic acid at 0 ppm. Unequivocal assignments of these four resonances in the spectrum of 5FW wtPAI-1 to specific tryptophan residues were accomplished by measuring the chemical shifts of the (19)F resonances of the single tryptophan mutants. There was close agreement between the resonances observed in 5FW wtPAI-1 and of those in the mutants for all three protein forms. This would imply little structural perturbation in the local structures of the tryptophan residues resulting from substitution by phenylalanine. The 5FW wtPAI-1 was observed to have lower second-order rate constant (k(app)) for the inhibition of t-PA than the natural tryptophan wtPAI-1, suggesting that the decreased activity may result from a small structural effect of the fluorine substituent of the indole ring. Further alterations in the k(app) and the stoichiometry of inhibition (SI) were observed in each of the mutants indicating an effect of the three tryptophan to phenylalanine mutations. Detailed interpretation of the (19)F NMR spectra of the PAI-1 mutants provides insights into the local segmental structure of the active form of the proteins and the structural changes that occur in the cleaved and t-PA complexed forms.     

Hydrophobicity of amino acid residues: differential scanning calorimetry and synthesis of the aromatic analogues of the polypentapeptide of elastin.

Relative hydrophobicities of aromatic amino acid residues are investigated by using differential scanning calorimetry (DSC) on 10 synthetic copolypentapeptides of poly(VPGVG) of elastin. Utilizing the hydrophobic-driven process of the inverse temperature transition exhibited by these polypentapeptides in aqueous solution, the relative hydrophobicities of Phe, Trp, and Tyr residues are determined by the critical temperature and heat of the transition. The DSC data for the aromatic residue containing copolypentapeptide aqueous solution indicate that tryptophan is the most hydrophobic amino acid residue, phenylalanine the third most hydrophobic on basis of transition temperature and the second on basis of transition heat. For tyrosine, significant differences are observed between the phenolic and the phenoxide anionic states. At pH 7, where tyrosine is protonated, it is found to be the second most hydrophobic amino acid residue on the basis of the transition temperature, whereas on the basis of the heat of transition, it is less hydrophobic than both tryptophan and phenylalanine. Changing the pH from pH 7 to pH 12, for example, for poly[0.8(VPGVG), 0.2(VPGYG)] in aqueous solution shifts the transition temperature from 7 to 49 degrees C with a dramatically reduced heat. On the basis of both the transition temperature scale and the heat of transition, the hydroxylated tyrosine appears less hydrophobic than glycine.