Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO^® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.     

Exciton circular dichroism couplet arising from nitrile-derivatized aromatic residues as a structural probe of proteins

Exciton coupling between two chromophores can produce a circular dichroism (CD) couplet that depends on their separation distance, among other factors. Therefore, exciton CD signals arising from aromatic sidechains, especially those of tryptophan (Trp), have been used in various protein conformational studies. However, the long-wavelength component of the commonly used CD couplet produced by a pair of Trp residues is typically located around 230 nm, thereby overlapping significantly with the protein backbone CD signal. This overlap often prevents a direct and quantitative assessment of the Trp CD couplet in question without further spectral analysis. Here, we show that this inconvenience can be alleviated by using a derivative of Trp, 5-cyanotryptophan (Trp_CN), as the chromophore. Specifically, through studying a series of peptides that fold into either α-helical or ß-hairpin conformations, we demonstrate that in comparison with the Trp CD couplet, that arising from two Trp_CN residues not only is significantly red-shifted but also becomes more intense due to the larger extinction coefficient of the underlying electronic transition. In addition, we show that a pair of p-cyanophenylalanines (Phe_CN) or a Phe_CN–Trp_CN pair can also produce a distinct exciton CD couplet that can be useful in monitoring conformational changes in proteins.     

Interactions of superoxide anion with enzyme radicals: Kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer

The kinetics of O^·-₂ reaction with semi-oxidized tryptophan radicals in lysozyme, Trp^·(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp^·(Lyz) radicals were formed by Br^·-₂ oxidation of the 3–4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 10⁸M^-1s^-1; but drops to 8 × 10⁷M^-1s^-1 or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp^·(Lyz) + Trp^·(Lyz) recombination reaction is about (4-7 × 10⁶M^-1s^-1) at/or below pH 6.2 then drops to 1.3-1.6 × 10⁶M^-1s^-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp^· residues upon Br^·-₂ reaction. The kinetics of Trp^·(Lyz) with ascorbate, a reducing species rather larger than O^·-₂ have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals-leading to the repair of the semi-oxidized Trp^·(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp^·(Lyz)+O^·-₂ reaction, as is most of the Trp^·(Lyz)+Trp^·(Lyz) reaction. However, the kinetic behavior of Trp^·(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O^·-₂. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O^·-₂ reactivity. To our knowledge, this is the first report on the kinetics of a protein-O^·-₂ reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.     

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3-Butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l-BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue¹⁹². Site-directed mutagenesis of three active site residues of meso-BDH, including Trp¹⁹⁰, which corresponds to Trp¹⁹² of l-BDH, converted its stereospecificity to that of l-BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.     

Charged or Aromatic Anchor Residue Dependence of Transmembrane Peptide Tilt

The membrane-spanning segments of integral membrane proteins often are flanked by aromatic or charged amino acid residues, which may “anchor” the transmembrane orientation. Single spanning transmembrane peptides such as those of the WALP family, acetyl-GWW(LA)_nLWWA-amide, furthermore adopt a moderate average tilt within lipid bilayer membranes. To understand the anchor residue dependence of the tilt, we introduce Leu-Ala “spacers” between paired anchors and in some cases replace the outer tryptophans. The resulting peptides, acetyl-GX²ALW(LA)₆LWLAX²²A-amide, have Trp, Lys, Arg, or Gly in the two X positions. The apparent average orientations of the core helical sequences were determined in oriented phosphatidylcholine bilayer membranes of varying thickness using solid-state ²H NMR spectroscopy. When X is Lys, Arg, or Gly, the direction of the tilt is essentially constant in different lipids and presumably is dictated by the tryptophans (Trp⁵ and Trp¹⁹) that flank the inner helical core. The Leu-Ala spacers are no longer helical. The magnitude of the apparent helix tilt furthermore scales nicely with the bilayer thickness except when X is Trp. When X is Trp, the direction of tilt is less well defined in each phosphatidylcholine bilayer and varies up to 70° among 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine bilayer membranes. Indeed, the X = Trp case parallels earlier observations in which WALP family peptides having multiple Trp anchors show little dependence of the apparent tilt magnitude on bilayer thickness. The results shed new light on the interactions of arginine, lysine, tryptophan, and even glycine at lipid bilayer membrane interfaces.     

Multidimensional1H and15N NMR investigation of glutamine-binding protein ofEscherichia coli

Summary Specific and uniform¹⁵N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His¹⁵⁶, Trp³² and Trp.²²⁰ residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport ofl-glutamine across the cytoplasmic membrane ofEscherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H₂ of His¹⁵⁶ refines the earlier, model where this particular proton formas an intermolecular hydrogen bond to the -carbonyl ofl-glutamine, while assignments of both Trp³² and Trp²²⁰ show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8–9 amino acid residues at a time. This paper illustrates the usefulness of combining¹⁵N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25 000.     

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.     

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D)¹H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs ɛ-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His³, Trp⁷², Tyr⁴¹, Tyr⁵⁰ and Tyr⁷⁴) appear to be exposed and accessible to 3-N-car☐ymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp²⁵ and Trp⁶² indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp⁷² ring in the presence of BASA. Moreover, a number of H^β resonances can be identified and sorted according to specific types of amino acid residues.     

Intra and intermolecular charge effects on the reaction of the superoxide radical anion with semi-oxidized tryptophan in peptides and N-acetyl tryptophan

The reaction of the superoxide radical anion (O^-·₂), with the semi-oxidized tryptophan neutral radical (Trp^·) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4–5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O^-·₂ with the Trp^· radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp^· radical formed from NAT with O^-·₂ is reduced to half that of the free Trp^· in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp^· and O^-·₂ may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.     

The Ionic and Hydrophobic Interactions Are Required for the Auto Activation of Cysteine Proteases of Plasmodium falciparum

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are major hemoglobinases and potential antimalarial drug targets. Our previous studies demonstrated that these enzymes are equipped with specific domains for specific functions. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. As with many proteases, falcipain-2 and falcipain-3 are synthesized as inactive zymogens. However, it is not known how these enzymes get activated for hemoglobin hydrolysis. In this study, we are presenting the first evidence that salt bridges and hydrophobic interactions are required for the auto activation of cysteine proteases of P.falciparum. To investigate the mechanism of activation of these enzymes, we expressed the wild type protein as well as different mutants in E.coli. Refolding was assessed by circular dichroism. Both CD and trans activation data showed that the wild type enzymes and mutants are rich in secondary structures with similar folds. Our study revealed that prodomain-mature domain of falcipain-2 and falcipain-3 interacts via salt bridges and hydrophobic interactions. We mutated specific residues of falcipain-2 and falcipain-3, and evaluated their ability to undergo auto processing. Mutagenesis result showed that two salt bridges (Arg ¹⁸⁵ - Glu ²²¹, Glu ²¹⁰ - Lys ⁴⁰³) in falcipain-2, and one salt bridge (Arg ²⁰²-Glu ²³⁸) in falcipain-3, play crucial roles in the activation of these enzymes. Further study revealed that hydrophobic interactions present both in falcipain-2 (Phe^214, Trp⁴⁴⁹ Trp ⁴⁵³) and falcipain-3 (Phe ²³¹ Trp ⁴⁵⁷ Trp ⁴⁶¹) also play important roles in the activation of these enzymes. Our results revealed the interactions involved in auto processing of two major hemoglobinases of malaria parasite.     

Mutations in transhydrogenase change the fluorescence emission state of TRP72 from 1La to 1Lb

The dI component of Rhodospirillum rubrum transhydrogenase has a single Trp residue (Trp⁷²), which has distinctive optical properties, including short-wavelength fluorescence emission with clear vibrational fine structure, and long-lived, well-resolved phosphorescence emission. We have made a set of mutant dI proteins in which residues contacting Trp⁷² are conservatively substituted. The room-temperature fluorescence-emission spectra of our three Met⁹⁷ mutants are blue shifted by ∼4 nm, giving them a shorter-wavelength emission than any other protein described in the literature, including azurin from Pseudomonas aeruginosa. Fluorescence spectra in low-temperature glasses show equivalent well-resolved vibrational bands in wild-type and the mutant dI proteins, and in azurin. Substitution of Met⁹⁷ in dI changes the relative intensities of some of these vibrational bands. The analysis supports the view that fluorescence from the Met⁹⁷ mutants arises predominantly from the ¹L_b excited singlet state of Trp⁷², whereas ¹L_a is the predominant emitting state in wild-type dI. It is suggested that the sulfur atom of Met⁹⁷ promotes greater stabilization of ¹L_a than either ¹L_b or the ground state. The phosphorescence spectra of Met⁹⁷ mutants are also blue-shifted, indicating that the sulfur atom decreases the transition energy between the ³L_a state of the Trp and the ground state.     

Toxin III of the scorpionAndroctonus australis Hector: Proton nuclear magnetic resonance assignments and secondary structure

Summary ¹H NMR has been applied to a3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpionAndroctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20–28) which is linked by two disulfide bridges to the central strand of a triple-stranded antiparallel -sheet. Turns were identified at residues 15–17, 47–49 and also at residues 51–53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu³⁷, Leu²³, Val⁴⁷, Tyr¹⁴, Trp⁴⁵ and Tyr⁵. The Trp⁴⁵ and Tyr⁵ rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.Thenomenclature used is similar to that described by Wütrich, 1986.     

Production of lysozyme and lysozyme-superoxide dismutase dimers bound by a ditryptophan cross-link in carbonate radical-treated lysozyme

Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp²⁸ and Trp¹²³ and N-formylkynurenine at Trp²⁸, Trp⁶² and Trp¹²³. Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp²⁸. The dimers were characterized as lysozyme-Trp²⁸-Trp²⁸-lysozyme and lysozyme-Trp²⁸-Trp³²-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed.     

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel,hHV1

Part of the “signature sequence” that defines the voltage-gated proton channel (H_V1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H_V1 genes. Replacing Trp²⁰⁷ in human H_V1 (hH_V1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, E_a, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp²⁰⁷ establishes the major energy barrier between closed and open hH_V1. Cation–π interaction between Trp²⁰⁷ and Arg²¹¹ evidently latches the channel closed. Trp²⁰⁷ mutants lost proton selectivity at pH_o >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H_V1 that is essential to its biological functions, was compromised. In the WT hH_V1, ΔpH-dependent gating is shown to saturate above pH_i or pH_o 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp²⁰⁷ mutants, ΔpH-dependent gating saturated at lower pH_o but not at lower pH_i. That Trp²⁰⁷ mutation selectively alters pH_o sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H_V1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH_o and pH_i in H_V1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.     

New insight into the binding mode of peptides at urotensin‐II receptor by Trp‐constrained analogues of P5U and urantide

Urotensin II (U‐II) is a disulfide bridged peptide hormone identified as the ligand of a G‐protein‐coupled receptor. Human U‐II (H‐Glu‐Thr‐Pro‐Asp‐c[Cys‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U‐II termed P5U (H‐Asp‐c[Pen‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) and the compound termed urantide (H‐Asp‐c[Pen‐Phe‐d ‐Trp‐Orn‐Tyr‐Cys]‐Val‐OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp⁷ residue was replaced by the highly constrained l ‐Tpi and d ‐Tpi residues. The replacement of the Trp⁷ by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation–activity relationships previously reported on UT receptor ligands. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of Short- and Long-Range Interactions on Trp Rotamer Populations Determined by Site-Directed Tryptophan Fluorescence of Tear Lipocalin

In the lipocalin family, the conserved interaction between the main α-helix and the β-strand H is an ideal model to study protein side chain dynamics. Site-directed tryptophan fluorescence (SDTF) has successfully elucidated tryptophan rotamers at positions along the main alpha helical segment of tear lipocalin (TL). The rotamers assigned by fluorescent lifetimes of Trp residues corroborate the restriction expected based on secondary structure. Steric conflict constrains Trp residues to two (t, g ⁻) of three possible χ₁ (t, g ⁻, g ⁺) canonical rotamers. In this study, investigation focused on the interplay between rotamers for a single amino acid position, Trp 130 on the α-helix and amino acids Val 113 and Leu 115 on the H strand, i.e. long range interactions. Trp130 was substituted for Phe by point mutation (F130W). Mutations at positions 113 and 115 with combinations of Gly, Ala, Phe residues alter the rotamer distribution of Trp130. Mutations, which do not distort local structure, retain two rotamers (two lifetimes) populated in varying proportions. Replacement of either long range partner with a small amino acid, V113A or L115A, eliminates the dominance of the t rotamer. However, a mutation that distorts local structure around Trp130 adds a third fluorescence lifetime component. The results indicate that the energetics of long-range interactions with Trp 130 further tune rotamer populations. Diminished interactions, evident in W130G113A115, result in about a 22% increase of α-helix content. The data support a hierarchic model of protein folding. Initially the secondary structure is formed by short-range interactions. TL has non-native α-helix intermediates at this stage. Then, the long-range interactions produce the native fold, in which TL shows α-helix to β-sheet transitions. The SDTF method is a valuable tool to assess long-range interaction energies through rotamer distribution as well as the characterization of low-populated rotameric states of functionally important excited protein states.     

Characterization of two fluorescent tryptophans in recombinant human granulocyte-colony stimulating factor: Comparison of native sequence protein and tryptophan-deficient mutants

In order to probe the role of the individual tryptophans of granulocyte-colony stimulating factor (G-CSF) inpH and guanidine HCl-induced fluorescence changes, site-directed mutagenesis was used to generate mutants replacing Trp¹¹⁸, Trp⁵⁸, or both with phenylalanine. Neither Trp to Phe mutation affected the folding or activity of the recombinant G-CSF, and the material expressed in yeast behaved identically to that expressed inEscherichia coli. All of the G-CSF species responded topH and guanidine HCl in qualitatively the same manner. Trp⁵⁸ has a fluorescence maximum at 350 nm and is quenched to a greater extent by the addition of guanidine HCl, indicating that it is fully solvent-exposed. Trp¹¹⁸ has a fluorescence maximum at 344 nm, and is less solvent-accessible than Trp⁵⁸. The analog in which both tryptophans have been replaced with phenylalanine shows only tyrosine fluorescence, with a peak at 304 nm which decreases with increasingpH. The intensity of the tyrosine fluorescence in this analog is much greater than that of the native sequence protein or single tryptophan mutants, indicating that energy transfer is taking place from tyrosine to tryptophan in these molecules. Below neutralpH the tyrosine fluorescence is much greater in the [Phe⁵⁸]G-CSF than in the [Phe¹¹⁸]G-CSF, indicating that Trp⁵⁸ might be a more efficient recipient of energy transfer from the tyrosine(s).     

Position—Specific contribution of interface tryptophans on membrane protein energetics

Interface tryptophans are key residues that facilitate the folding and stability of membrane proteins. Escherichia coli OmpX possesses two unique interface tryptophans, namely Trp76, which is present at the interface and is solvent-exposed, and Trp140, which is relatively more lipid solvated than Trp76 in symmetric lipid membranes. Here, we address the requirement for tryptophan and the consequences of aromatic amino acid substitutions on the folding and stability of OmpX. Using spectroscopic measurements of OmpX-Trp/Tyr/Phe mutants, we show that the specific mutation W76 → Y allows barrel assembly > 1.5-fold faster than native OmpX, and increases stability by ~ 0.4 kcal mol^− 1. In contrast, mutating W140 → F/Y lowers OmpX thermodynamic stability by ~ 0.4 kcal mol^− 1, without affecting the folding kinetics. We conclude that the stabilizing effect of tryptophan at the membrane interface can be position—and local environment—specific. We propose that the thermodynamic contributions for interface residues be interpreted with caution.     

Kringle 4 from human plasminogen:1H-nuclear magnetic resonance study of the interactions between ω-amino acid ligands and aromatic residues at the lysine-binding site

The interactions of theω-amino acid ligandsε-aminocaproic acid andp-benzylaminesulphonic acid with the isolated kringle 4 domain from human plasminogen have been investigated by¹H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Overall, the data indicate that binding either ligand does not cause the kringle to undergo significant conformational changes. When p-benzylaminesulphonic acid is in excess relative to the kringles, progressive exchange-broadening and high field chemical shifts are observed for the proton resonances of the ligand. The largest effect is seen at the amino end of the molecule, which indicates that the — NH ₃ ⁺ group of the ligand penetrates deeper into the binding site than does the — SO ₃ ^- . Ligand-binding causes signals from the ring-current shifted Leu⁴⁶ CH ₃ ^δ .δ groups and from a number of aromatic side-chains to shift. Depending on the ligand, the latter include Tyr-II (Tyr⁵⁰), Tyr-V (an immobile ring), His-II and His-III imidazole groups and the three Trp indole groups present in kringle 4. In particular,p-benzylaminesulphonic acid-binding induces large high field shifts on the Trp-II H6 triplet and the Trp-III (Trp⁷²) H2 singlet. On the other hand,ε-aminocaproic acid bound to kringle 4 exhibits large chemical shifts of its CH₂ proton resonances, which indicates that the lysine-binding site is rich in aromatic side chains. Overhauser experiments centered on thep-benzylaminesulphonic acid H2,6 and H3,5 aromatic transitions as well as on the shifted Trp-II and Trp-III signals reveal efficient cross-relaxation between these two indole side chains and thep-benzylaminesulphonic acid ring. These experiments also show that the side chains from Phe⁶⁴, Tyr-II (Tyr⁵⁰), Tyr-IV, and His-II (His³¹) interact with the ligand. In combination with reported chemical modification experiments that show requirement of Asp⁵⁷, Arg⁷¹ and Trp⁷² integrity for ligand-binding, our study underscores the relevance of the Cys⁵¹-Cys⁷⁵ loop in defining the kringles’ lysine-binding site. Furthermore, the Cys²²-Cys⁶³ loop is folded so as to place His³¹, His³³, Tyr⁴¹ and Leu⁴⁶ in proximity to the binding site. The involvement of residues within the Cys⁵¹-Cys⁷⁵ loop in ligand-binding suggests that Trp-II and Tyr-IV may correspond to Trp⁶² and Tyr⁷⁴, respectively. As shown by Overhauser experiments, these two residues are in close contact with each other. From these studies and from the shielding and deshielding effects caused byp-benzylaminesulphonic acid, we suggest that the ligand is sandwiched between the indole rings of Trp-II and Trp-III, which form part of the hydrophobic binding site.