Solvent-exposed Trp Research Articles

Magnetic-Field Dependence of LC-Photo-CIDNP in the Presence of Target Molecules Carrying a Quasi-Isolated Spin Pair.

NMR spectroscopy is well known for its superb resolution, especially at high applied magnetic field. However, the sensitivity of this technique is very low. Liquid-state low-concentration photo-chemically-induced dynamic nuclear polarization (LC-photo-CIDNP) is a promising emerging methodology capable of enhancing NMR sensitivity in solution. LC-photo-CIDNP works well on solvent-exposed Trp and Tyr residues, either in isolation or within proteins. This study explores the magnetic-field dependence of the LC-photo-CIDNP experienced by two tryptophan isotopologs in solution upon in situ LED-mediated optical irradiation. Out of the two uniformly 13C,15N-labeled Trp (Trp-U-13C,15N) and Trp-α-13C-β,β,2,4,5,6,7-d7 species employed here, only the latter bears a quasi-isolated 1Hα-13Cα spin pair. Computer simulations of the predicted polarization due to geminate recombination of both species display a roughly bell-shaped field dependence. However, while Trp-U-13C,15N is predicted to show a maximum at ca. 500 MHz (11.7 T) and a fairly weak field dependence, Trp-α-13C-β,β,2,4,5,6,7-d7 is expected to display a much sharper field dependence accompanied by a dramatic polarization increase at lower field (ca. 200 MHz, 4.7 T). Experimental LC-photo-CIDNP studies on both Trp isotopologs at 1μM concentration, performed at selected fields, are consistent with the theoretical predictions. In summary, this study highlights the prominent field-dependence of LC-photo-CIDNP enhancements () experienced by Trp isotopologs bearing a quasi-isolated spin pair.

PH-induced structural change of a multi-tryptophan protein MPT63 with immunoglobulin-like fold: identification of perturbed tryptophan residue/residues

The structural change of M. tuberculosis MPT63, which is predominantly a β-sheet protein having an immunoglobulin like fold, has been investigated in the pH range 7.5–1.5 using various biophysical techniques along with low-temperature phosphorescence (LTP) spectroscopy. MPT63 contains four Tryptophan (Trp) residues at 26, 48, 82, and 129. Although circular dichroism, steady-state and time-resolved fluorescence, time-resolved anisotropy, 1-aniline-8-naphthalene sulfonic (ANS) acid binding, and analytical ultracentrifuge depict more open largely unfolded structure of MPT63 at pH 1.5 and also more accessible nature of Trp residues to neutral quencher at pH 1.5, it is, however, not possible to assign the specific Trp residue/residues being perturbed. This problem has been resolved using LTP of MPT63, which shows optically resolved four distinct (0, 0) bands corresponding to four Trp residues in the pH range 7.5–3.0. LTP at pH 1.5 clearly reveals that the solvent-exposed Trp 82 and the almost buried Trp 129 are specifically affected compared with Trp 48 and Trp 26. Lys 8 and Lys 27 are predicted to affect Trp 129. Tyrosine residues are found to be silent even at pH 1.5. This type of specific perturbation in a multi-Trp protein has not been addressed before. LTP further indicates that partially exposed Trp 48 is preferentially quenched by acrylamide compared with other Trp residues at both pH 7.5 and 1.5. The solvent-exposed Trp 82 is surprisingly found to be not quenched by acrylamide at pH 7.5. All the results are obtained using micromolar concentration of protein and without using any Trp-substituted mutant.

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1(WT) and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp(32) residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp(32) residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1(WT) and hSOD1(G93A) mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp(32) residue in the process. The results showed that Trp(32) residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp(32) residue (bovine SOD1 and hSOD1(W32F) mutant). The results support a role for the oxidation products of the hSOD1-Trp(32) residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed Trp 32 residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H 2O and H 2 18O, and analyzed by UV–Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp 32 residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp 32, partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo.

Understanding Wavelength Dependence of Tryptophan Fluorescence Decays

Understanding the ubiquitous non-exponential decay exhibited by tryptophan (Trp) fluorescence in proteins is becoming crucial for interpretation of ultrafast decay experiments, especially when the cause of time dependent Stokes shifts may be interpreted equally well by water/protein relaxation or by excited state decay of short-lived blue shifted subpopulations as a result of a heterogeneous microenvironment. Here we report hybrid quantum mechanical-molecular mechanical (QM-MM) simulations of fluorescence wavelengths and lifetimes for a set of 7 cyclic hexapeptides with rigid peptide backbone, a single Trp residue, and 5 other amino acids that do not quench Trp fluorescence in aqueous solution. These were designed specifically to examine possible fluorescence lifetime heterogeneity arising only from different rates of quenching by electron transfer (ET) from the excited indole ring to a nearby backbone amide by different Trp sidechain rotamers-- in the absence of nanosecond solvent relaxation. The results capture most of the unexpected diversity in observed spectroscopic properties of the 7 peptides, including the almost universal pronounced correlation of blue shifted decay associated spectra to have the shortest lifetimes. Heretofore no attractive physical mechanism has been advanced to explain why rotamers with blue shifted spectra should almost always have the shortest lifetime component. The familiar broad fluorescence spectrum of a solvent-exposed Trp with emission peak near 350 nm is found to be an ensemble average of single molecular fluorescence peaks fluctuating on a femtosecond time scale over 3000-4000 wavenumbers (fwhw) or 40 nm. This leads naturally to a picture in which those rotamers having shorter wavelength emission spectra (higher average energy) tend to have short lifetimes due to their increased probability for transient fast quenching during the large fluctuations in environment that bring the CT and 1La states into resonance. This mechanism should be operative in proteins.

1H Photo-CIDNP Enhancements in Heteronuclear Correlation NMR Spectroscopy

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is usually employed as a probe of solvent exposure in biomolecular NMR. The potential of the photo-CIDNP effect for sensitivity enhancement, however, remains poorly explored. Here, we introduce (1)H-photo-CIDNP in heteronuclear correlation spectroscopy at low laser irradiation power (1 W), and compare the sensitivity of various (1)H-photo-CIDNP-enhanced- (HPE) (1)H-(15)N heteronuclear correlation pulse sequences, including HSQC, HMQC, and SOFAST-HMQC, in terms of their ability to detect the Trp indole H(epsilon1) resonance. Both Trp and the Trp-containing protein apoHmpH were analyzed using flavin mononucleotide as photosensitizer in aqueous solutions either containing or lacking urea. We find that (1)H-(15)N photo-CIDNP-SOFAST-HMQC, denoted here as HPE-SOFAST-HMQC, yields a 2-fold higher signal-to-noise per unit time than the parent SOFAST-HMQC, for the solvent-exposed Trp of urea-unfolded apoHmpH. Thus, HPE-SOFAST-HMQC is the most sensitive heteronuclear correlation pulse sequence for the detection of solvent-exposed Trp.

Loop-Tryptophan Human Purine Nucleoside Phosphorylase Reveals Submillisecond Protein Dynamics

Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The catalytic sites of PNP are located near the subunit-subunit interfaces where F159 is a catalytic site residue donated from an adjacent subunit. F159 covers the top (beta) surface of the ribosyl group at the catalytic site. QM/MM calculations of human PNP have shown that F159 is the center of the most mobile region of the protein providing access to the substrate in the active site. F159 is also the key residue in a cluster of hydrophobic residues that shield catalytic site ligands from bulk solvent. Trp-free human PNP (Leuko-PNP) was previously engineered by replacing the three Trp residues of native PNP with Tyr. From this active construct, a single Trp residue was placed in the catalytic site loop (F159W-Leuko-PNP) as a reporter group for the ribosyl region of the catalytic site. The F159W-Leuko-PNP fluorescence is red shifted compared to native PNP, suggesting a solvent-exposed Trp residue. Upon ligand binding (hypoxanthine), the 3-fold fluorescence quench confirms conformational packing of the catalytic site pocket hydrophobic cluster. F159W-Leuko-PNP has an on-enzyme thermodynamic equilibrium constant (Keq) near unity in the temperature range between 20 and 30 degrees C and nonzero enthalpic components, making it suitable for laser-induced T-jump analyses. T-jump relaxation kinetics of F159W-Leuko-PNP in equilibrium with substrates and/or products indicate the conformational equilibria of at least two ternary complex intermediates in the nano- to millisecond time scale (1000-10000 s-1) that equilibrate prior to the slower chemical step (approximately 200 s-1). F159W-Leuko-PNP provides a novel protein platform to investigate the protein conformational dynamics occurring prior to transition state formation.

Electron Hopping through the 15 Å Triple Tryptophan Molecular Wire in DNA Photolyase Occurs within 30 ps

DNA photolyase is a photoactive flavoprotein that contains three tryptophan residues between the FAD cofactor and the protein surface, the solvent-exposed Trp being located 14.8 A from the flavin. Photoreduction of the neutral radical FADH. form to the catalytically active FADH- form occurs via electron transfer through this chain. The first step in this chain takes 30 ps, the second less than 4 ps. Using a combination of site-directed mutagenesis and femtosecond polarization spectroscopy to discriminate the spectroscopically indistinguishable Trp residues, we show that the third step occurs in less than 30 ps. This implies that the first photoreduction step is rate limiting and that the Trp chain effectively acts as molecular "wire" ensuring rapid and directed long-range charge translocation across the protein. This finding is important for the functioning of the large class of cryptochrome blue-light receptors, where the Trp chain is conserved. In DNA photolyase we make use of the natural photoactivation of the process, but more generally chains of aromatic amino acids may allow very fast long-range electron transfer also in nonphotoactive proteins.

Tertiary Structure and Carbohydrate Recognition by the Chitin-Binding Domain of a Hyperthermophilic Chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein–carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.

Multiple Tryptophan Probes Reveal that Ubiquitin Folds via a Late Misfolded Intermediate

Much of our understanding of protein folding mechanisms is derived from experiments using intrinsic fluorescence of natural or genetically inserted tryptophan (Trp) residues to monitor protein refolding and site-directed mutagenesis to determine the energetic role of amino acids in the native (N), intermediate (I) or transition (T) states. However, this strategy has limited use to study complex folding reactions because a single fluorescence probe may not detect all low-energy folding intermediates. To overcome this limitation, we suggest that protein refolding should be monitored with different solvent-exposed Trp probes. Here, we demonstrate the utility of this approach by investigating the controversial folding mechanism of ubiquitin (Ub) using Trp probes located at residue positions 1, 28, 45, 57, and 66. We first show that these Trp are structurally sensitive and minimally perturbing fluorescent probes for monitoring folding/unfolding of the protein. Using a conventional stopped-flow instrument, we show that ANS and Trp fluorescence detect two distinct transitions during the refolding of all five Trp mutants at low concentrations of denaturant: T1, a denaturant-dependent transition and T2, a slower transition, largely denaturant-independent. Surprisingly, some Trp mutants (UbM1W, UbS57W) display Trp fluorescence changes during T1 that are distinct from the expected U→N transition suggesting that the denaturant-dependent refolding transition of Ub is not a U→N transition but represents the formation of a structurally distinct I-state (U→I). Alternatively, this U→I transition could be also clearly distinguished by using a combination of two Trp mutations UbF45W-T66W for which the two Trp probes that display fluorescence changes of opposite sign during T1 and T2 (UbF45W-T66W). Global fitting of the folding/unfolding kinetic parameters and additional folding-unfolding double-jump experiments performed on UbM1W, a mutant with enhanced fluorescence in the I-state, demonstrate that the I-state is stable, compact, misfolded, and on-pathway. These results illustrate how transient low-energy I-states can be characterized efficiently in complex refolding reactions using multiple Trp probes.

Cation−π Interaction in Model α-Helical Peptides

Cation-pi interactions are increasingly recognized as important in chemistry and biology. Here we investigate the cation-pi interaction by determining its effect on the helicity of model peptides using a combination of CD and NMR spectroscopy. The data show that a single Trp/Arg interaction on the surface of a peptide can make a significant net favorable free energy contribution to helix stability if the two residues are positioned with appropriate spacing and orientation. The solvent-exposed Trp-->Arg (i, i + 4) interaction in helices can contribute -0.4 kcal/mol to the helix stability, while no free energy gain is detected if the two residues have the reversed orientation, Arg-->Trp (i, i + 4). The derived free energy is consistent with other experimental results studied in proteins or model peptides on cation-pi interactions. However in the same system the postulated Phe/Arg (i, i + 4) cation-pi interaction provides no net free energy to helix stability. Thus the Trp-->Arg interaction is stronger than Phe-->Arg. The cation-pi interactions are not sensitive to the screening effect by adding neutral salt as indicated by salt titration. Our results are in qualitative agreement with theoretical calculations emphasizing that cation-pi interactions can contribute significantly to protein stability with the order Trp > Phe. However, our and other experimental values are significantly smaller than estimates from theoretical calculations.

Uterocalin, a lipocalin provisioning the preattachment equine conceptus: fatty acid and retinol binding properties, and structural characterization.

The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and fluorescence-based assays, we show that equine uterocalin binds the fluorescent fatty acids 11-(dansylamino)undecanoic acid, dansyl-D,L-alpha-amino-octanoic acid and cis-parinaric acid, and, by competition, oleic, palmitic, arachidonic, docosahexaenoic, gamma-linolenic, cis-eicosapentaenoic and linoleic acids. Uterocalin also binds all-trans-retinol, the binding site for which is coincident or interactive with that for fatty acids. Molecular modelling and intrinsic fluorescence analysis of the wild-type protein and a Trp-->Glu mutant protein indicated that uterocalin has an unusually solvent-exposed Trp side chain projecting from its large helix directly into solvent. This feature is unusual among lipocalins and might relate to binding to, and uptake by, the trophoblast. Uterocalin therefore has the localization and binding activities for the provisioning of the equine conceptus with lipids including those essential for morphogenesis and pattern formation. The possession of a fibrous capsule surrounding the conceptus might be an ancestral condition in mammals; homologues of uterocalin might be essential for early development in marsupials and in eutherians in which there is a prolonged preimplantation period.

Uterocalin, a lipocalin provisioning the preattachment equine conceptus: fatty acid and retinol binding properties, and structural characterization

The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and fluorescence-based assays, we show that equine uterocalin binds the fluorescent fatty acids 11-(dansylamino)undecanoic acid, dansyl-d,l-α-amino-octanoic acid and cis-parinaric acid, and, by competition, oleic, palmitic, arachidonic, docosahexaenoic, γ-linolenic, cis-eicosapentaenoic and linoleic acids. Uterocalin also binds all-trans-retinol, the binding site for which is coincident or interactive with that for fatty acids. Molecular modelling and intrinsic fluorescence analysis of the wild-type protein and a Trp → Glu mutant protein indicated that uterocalin has an unusually solvent-exposed Trp side chain projecting from its large helix directly into solvent. This feature is unusual among lipocalins and might relate to binding to, and uptake by, the trophoblast. Uterocalin therefore has the localization and binding activities for the provisioning of the equine conceptus with lipids including those essential for morphogenesis and pattern formation. The possession of a fibrous capsule surrounding the conceptus might be an ancestral condition in mammals; homologues of uterocalin might be essential for early development in marsupials and in eutherians in which there is a prolonged preimplantation period.

Sj-FABPc fatty-acid-binding protein of the human blood fluke Schistosoma japonicum: structural and functional characterization and unusual solvent exposure of a portal-proximal tryptophan residue.

Sj-FABPc of the blood fluke of humans, Schistosoma japonicum, is a member of the FABP/P2/CRBP/CRABP family of beta-barrel cytosolic fatty-acid-binding and retinoid-binding proteins. Sj-FABPc has at least eight different variants encoded by a single-copy polymorphic gene. In fluorescence-based assays, recombinant Sj-FABPc was found to bind 11-(dansylamino)undecanoic acid (DAUDA), inducing a shift in peak fluorescence emission from 543 to 493 nm. A similar spectral change was observed in dansyl-amino-octanoic acid (in which the dansyl fluorophore is attached at the alpha-carbon rather than the omega-carbon of DAUDA), indicating that the ligand enters entirely into the binding site. Sj-FABPc also bound the naturally fluorescent cis-parinaric acid, as well as oleic acid and arachidonic acid, by competition, but not all-trans-retinol. Dissociation constants were, for cis-parinaric acid, K(d)=2.5+/-0.1 microM (mean+/-S.E.M.) and an apparent stoichiometry consistent with one binding site per molecule of Sj-FABPc and, for oleic acid, K(i) approximately 80 nM. A deletion mutant from which alpha-II was absent failed to bind ligand. Sj-FABPc modelled well to known structures of the protein family; an unusually solvent-exposed Trp side chain was evident adjacent to the presumptive portal through which ligand is thought to enter and leave. Intrinsic fluorescence analyses of Sj-FABPc and of the deletion mutant (from which Trp-27 is absent) confirmed the unusual disposition of this side chain. Virtually all members of the FABP/P2/CRBP/CRABP protein family have prominent hydrophobic side chains in this position, with the exception of liver FABP and ileal FABP, which instead have charged side chains. Liver FABP is known to be distinct from other members of the protein family in that it does not seem to contact membranes to collect and deposit its ligand. It is therefore postulated that the unusually positioned apolar side chains in Sj-FABPc and others in the family are important in interactions with membranes or other cellular components.

Sj-FABPc fatty-acid-binding protein of the human blood fluke Schistosoma japonicum: structural and functional characterization and unusual solvent exposure of a portal-proximal tryptophan residue

Sj-FABPc of the blood fluke of humans, Schistosoma japonicum, is a member of the FABP/P2/CRBP/CRABP family of β-barrel cytosolic fatty-acid-binding and retinoid-binding proteins. Sj-FABPc has at least eight different variants encoded by a single-copy polymorphic gene. In fluorescence-based assays, recombinant Sj-FABPc was found to bind 11-(dansylamino)undecanoic acid (DAUDA), inducing a shift in peak fluorescence emission from 543 to 493 nm. A similar spectral change was observed in dansyl-amino-octanoic acid (in which the dansyl fluorophore is attached at the α-carbon rather than the Ω-carbon of DAUDA), indicating that the ligand enters entirely into the binding site. Sj-FABPc also bound the naturally fluorescent cis-parinaric acid, as well as oleic acid and arachidonic acid, by competition, but not all-trans-retinol. Dissociation constants were, for cis-parinaric acid, Kd = 2.5±0.1 μM (mean±S.E.M.) and an apparent stoichiometry consistent with one binding site per molecule of Sj-FABPc and, for oleic acid, Ki≈ 80 nM. A deletion mutant from which α-II was absent failed to bind ligand. Sj-FABPc modelled well to known structures of the protein family; an unusually solvent-exposed Trp side chain was evident adjacent to the presumptive portal through which ligand is thought to enter and leave. Intrinsic fluorescence analyses of Sj-FABPc and of the deletion mutant (from which Trp-27 is absent) confirmed the unusual disposition of this side chain. Virtually all members of the FABP/P2/CRBP/CRABP protein family have prominent hydrophobic side chains in this position, with the exception of liver FABP and ileal FABP, which instead have charged side chains. Liver FABP is known to be distinct from other members of the protein family in that it does not seem to contact membranes to collect and deposit its ligand. It is therefore postulated that the unusually positioned apolar side chains in Sj-FABPc and others in the family are important in interactions with membranes or other cellular components.

Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity

The three-dimensional solution structure of circulin A, a 30 residue polypeptide from the African plant Chassalia parvifolia, has been determined using two-dimensional 1H-NMR spectroscopy. Circulin A was originally identified based upon its inhibition of the cytopathic effects and replication of the human immunodeficiency virus. Structural restraints consisting of 369 interproton distances inferred from nuclear Overhauser effects, and 21 backbone dihedral and nine χ1 angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimisation in the program X-PLOR. The final set of 12 structures had mean pairwise rms differences over the whole molecule of 0.91 Å for the backbone atom, and 1.68 Å for all heavy atoms. For the well-defined region encompassing residues 2–12 and 18–27, the corresponding values were 0.71 and 1.66 Å, respectively. Circulin A adopts a compact structure consisting of β-turns and a distorted segment of triple-stranded β-sheet. Fluorescence spectroscopy provided additional evidence for a solvent-exposed Trp residue. The molecule is stabilised by three disulfide bonds, two of which form an embedded loop completed by the backbone fragments connecting the cysteine residues. A third disulfide bond threads through the centre of this loop to form a “cystine-knot” motif. This motif is present in a range of other biologically active proteins, including ω-contoxin GVIA and Cucurbita maxima trypsin inhibitor. Circulin A belongs to a novel class of macrocyclic peptides which have been isolated from plants in the Rubiaceae family. The global fold of circulin A is similar to kalata B1, the only member of this class for which a structure has previously been determined.

Serpin alpha 1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy.

Various conformational forms of the archetypal serpin human alpha 1proteinase inhibitor (alpha 1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because alpha 1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluorescence quenching resolved spectra and red-edge excitation measurements of the Trp environment. Thus, it is possible to define the conformation of the various forms based on the observed fluorescent properties of each of the Trp residues measured under a range of conditions. We show that denaturation in GuHCl, or thermal denaturation in Tris, followed by renaturation, leads to the formation of polymers that contain solvent-exposed Trp 238, which we interpret as ordered head-to-tail polymers (A-sheet polymers). However, thermal denaturation in citrate leads to shorter polymers where some of the Trp 238 residues are not solvent accessible, which we interpret as polymers capped by head-to-head interactions via the C sheet. The latter treatment also generates monomers thought to represent a latent form, but in which the environment of Trp 238 is occluded by ionized groups. These data indicate that the folding pathway of alpha 1PI, and presumably other serpins, is sensitive to solvent composition that affects the affinity of the reactive site loop for the A sheet or the C sheet.

Comparative triplet-state properties of the three tryptophan residues in bacteriophage T4 lysozyme and in the enzyme complex with methylmercury(II).

Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane.

Structure of oxidized flavodoxin from Anacystis nidulans

The structure of oxidized flavodoxin from the cyanobacterium Anacystis nidulans has been determined at 2.5 A resolution with phases calculated from ethylmercury phosphate and dimercuriacetate derivatives. The determination of partial sequences, including a total of 85 residues, has assisted in the interpretation of the electron density. Preliminary refinement of a partial model (1072 atoms) has reduced R to 0.349 for the 10.997 reflections between 2.0 and 5.0 A with 1 greater than 2 sigma. The polypeptide backbone, which comprises 167 residues in the current model, adopts the familiar beta-alpha-beta conformation found in other flavodoxins and in the nucleotide-binding domains of the pyridine-nucleotide dehydrogenases, with five parallel strands in the central sheet. Comparison with flavodoxin from Clostridium MP (138 residues) shows that extra residues of A. nidulans flavodoxin are accommodated in a major insertion about 20 residues in length, which forms a lobe adjacent to the fifth strand of parallel sheet, and in additions to several external segments. Residues added between the fourth sheet strand and the start of the third helix alter the environment of the pyrimidine end of the flavin mononucleotide ring. The flavin mononucleotide phosphate binds to the start of helix 1, interacting with hydroxyamino acids and with main-chain amide groups. Two hydrophobic residues, both tentatively identified as Trp, enclose the isoalloxazine ring; the solvent-exposed Trp is nearly parallel to the flavin ring. The hydrophobic environment provided by these residues must be partly responsible for the pronounced vibrational resolution of the flavin spectrum near 450 nm. The flavin ring is tilted relative to its orientation in Clostridium MP flavodoxin. In addition, atoms N-3 and O-2 alpha of the isoalloxazine appear to form hydrogen bonds to the backbone at CO97 and NH99 in a conformation entirely different from that found in Clostridium MP flavodoxin but structurally analogous to Desulfovibrio vulgaris flavodoxin.