Urea-dependent unfolding of murine adenosine deaminase: sequential destabilization as measured by 19F NMR

Murine adenosine deaminase (mADA) is a 40 kDa (beta/alpha)(8)-barrel protein consisting of eight central beta-strands and eight peripheral alpha-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-(19)F-Trp). The (19)F NMR spectrum of 6-(19)F-Trp-labeled mADA reveals four distinct resonances in the native state and three partly overlapped resonances in the unfolded state. The resonances were assigned unambiguously by site-directed mutagenesis. Equilibrium unfolding of 6-(19)F-Trp-labeled mADA was monitored using (19)F NMR based on these assignments. The changes in intensity of folded and unfolded resonances as a function of urea concentration show transition midpoints consistent with data observed by far-UV CD and fluorescence spectroscopy, indicating that conformational changes in mADA during urea unfolding can be followed by (19)F NMR. Chemical shifts of the (19)F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-(19)F-Trp residues change differently. The urea-induced changes in local structure around four 6-(19)F-Trp residues of mADA were analyzed on the basis of the tertiary structure and chemical shifts of folded resonances. The results reveal that different local regions in mADA have different urea-dependent behavior, and that local regions of mADA change sequentially from native to intermediate topologies on the unfolding pathway.     

Comparison of C40/82A and P27A C40/82A barstar mutants using 19F NMR

Barstar, an inhibitor of the enzyme barnase, contains two phenylalanine residues, three tryptophan residues, and two proline residues. After incorporating either 2-19F-Phe, 4-19F-Phe, or 6-19F-Trp, the structural, dynamic, and folding properties of two mutants (C40/82A, a double mutant, and P27A C40/82A, a triple mutant) were studied by 19F NMR. Experiments were performed as a function of temperature and urea with the two mutants. We show that the consequences of the P27A mutation are extensive. The effect of the mutation is transmitted to distant residues (Phe56 and Trp53) as well as to a residue deeply buried in the hydrophobic core (Phe74). By incorporating 2-19F-Phe, it is shown that Phe56 undergoes a slow ring flipping on the NMR time scale in the triple mutant that is not observed in the double mutant. On the other hand, incorporating 4-19F-Phe shows that the P27A mutation has little effect along the Cbeta-Cgamma axis of Phe56. Labeling with 4-19F-Phe shows, from line broadening, that Phe74 experiences more dynamic motion than does Phe56 in both the double and triple mutant. After incorporating 6-19F-Trp, it is found that, in the triple mutant, Trp53 shows conformational heterogeneity at low temperature while Trp44, which is close to the P27A mutation, does not. At 20 degrees C, residual native-like structure was detected around Trp53 at high concentrations of denaturant. Barstar is cold denatured in the presence of urea. For the double mutant at temperatures below 15 degrees C, and in the presence of 2.5-3.5 M urea, the resonance for Phe74 broadens, and two peaks are observed at 5 degrees C indicative of an exchange process. From line-shape analysis, assuming a two-site conformational exchange, the rate constants as a function of temperature can be extracted. An Eyring plot is linear at 0 M urea but deviates from linearity below 20 degrees C in the presence of 2.5 or 3.5 M urea. The data as a function of urea suggest sequential events in the unfolding process.     

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.     

(19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution

The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.     

Calcium and praseodymium complexes in solution. 1H n.m.r. conformational study of the model tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine

The synthetic tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine (Ac-DVDA) is a model of the calcium binding site of proteins such as carp parvalbumin, thermolysin and calmodulin. 1H n.m.r. spectra of the tetrapeptide are presented and assigned for D2O and DMSO solutions to determine the conformational mobility. The resonance of the two aspartyl side chains could be completely analysed and the vicinal coupling (C alpha H-C beta H and NH-C alpha H) indicated that the free peptide has considerable conformational mobility. The Ca(II) complex generates a different 1H n.m.r. spectrum for the aspartyl resonances at neutral pH. The solution conformation of Pr(III) complex of Ac-DVDA has been investigated using induced chemical shifts. The observed trends in the magnitude of the shift ratios and the rotamer population suggest that the metal ion binds predominantly to both carboxylates of two aspartyl residues in a bidentate fashion. We discuss the consistency of the differentiated spectra for aspartyl residues in the complex with the stepwise binding of Ca2+ to the carrier.     

Conformational flexibility of PEP mutase

Previous work has indicated that PEP mutase catalyzes the rearrangement of phosphoenolpyruvate to phosphonopyruvate by a dissociative mechanism. The crystal structure of the mutase with Mg(II) and sulfopyruvate (a phosphonopyruvate analogue) bound showed that the substrate is anchored to the active site by the Mg(II), and shielded from solvent by a large loop (residues 115-133). Here, the crystal structures of wild-type and D58A mutases, in the apo state and in complex with Mg(II), are reported. In both unbound and Mg(II)-bound states, the active site is accessible to the solvent. The loop (residues 115-133), which in the enzyme-inhibitor complexes covers the active site cavity, is partially disordered or adopts a conformation that allows access to the cavity. In the apo state, the residues associated with Mg(II) binding are poised to accept the metal ion. When Mg(II) binds, the coordination is the same as that previously observed in the enzyme-Mg(II) sulfopyruvate complex, except that the coordination positions occupied by two ligand oxygen atoms are occupied by two water molecules. When the loop opens, three key active site residues are displaced from the active site, Lys120, Asn122, and Leu124. Lys120 mediates Mg(II) coordination. Asn122 and Leu124 surround the transferring phosphoryl group, and thus prevent substrate hydrolysis. Amino acid replacement of any one of these three loop residues results in a significant loss of catalytic activity. It is hypothesized that the loop serves to gate the mutase active site, interconverting between an open conformation that allows substrate binding and product release and a closed conformation that separates the reaction site from the solvent during catalysis.     

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.     

Spectroscopic studies on cobalt(II)-substituted metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria

In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. (1)H NMR studies of Co(II)(1)-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)(1)-ImiS. The addition of a second Co(II) to Co(II)(1)-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn(2) site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn(1) site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.     

Calcium(II) site specificity: effect of size and charge on metal ion binding to an EF-hand-like site

The molecular mechanisms by which protein Ca(II) sites selectively bind Ca(II) even in the presence of high concentrations of other metals, particularly Na(I), K(I), and Mg(II), have not been fully described. The single Ca(II) site of the Escherichia coli receptor for D-galactose and D-glucose (GGR) is structurally related to the eukaryotic EF-hand Ca(II) sites and is ideally suited as a model for understanding the structural and electrostatic basis of Ca(II) specificity. Metal binding to the bacterial site was monitored by a Tb(III) phosphorescence assay: Ca(II) in the site was replaced with Tb(III), which was then selectively excited by energy transfer from protein tryptophans. Photons emitted from the bound Tb(III) enabled specific detection of this substrate; for other metals binding was detected by competitive displacement of Tb(III). Representative spherical metal ions from groups IA, IIA, and IIIA and the lanthanides were chosen to study the effects of metal ion size and charge on the affinity of metal binding. A dissociation constant was measured for each metal, yielding a range of KD's spanning over 6 orders of magnitude. Monovalent metal ions of group IA exhibited very low affinities. Divalent group IIA metal ions exhibited affinities related to their size, with optimal binding at an effective ionic radius between those of Mg(II) (0.81 A) and Ca(II) (1.06 A). Trivalent metal ions of group IIIA and the lanthanides also exhibited size-dependent affinities, with an optimal effective ionic radius between those of Sc(III) (0.81 A) and Yb(III) (0.925 A). The results indicate that the GGR site selects metal ions on the basis of both charge and size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton magnetic resonance studies on peptide fragments of troponin-C containing single calcium-binding sites

Proton magnetic resonance spectroscopy has been employed to study the solution conformation of three cleavage fragments of troponin-C, each containing a single Ca(II)-binding site and corresponding to different regions in the primary sequence; viz. CB8 (residues 46–77), CB9 (residues 85–134) and TH2 (residues 121–159). Although all three peptides lack a well-defined tertiary fold in the absence of metal ions, several spectral features indicate the presence of local conformational constraints in each apopeptide. Ca(II) binding led to spectral changes consistent with increased restriction of backbone motility and the adoption of a more compact conformation. Studies using paramagnetic ions as conformational probes support current views concerning the nature of the ligands at the metal binding sites.The nature and kinetics of the structural influence of metal binding suggest that the conformational constraints existing in the CB8 apo-peptide provide an adequate Ca(II)-binding configuration. In contrast, the CB9 and TH2 peptides exhibit spectral changes consistent with an increased local structure in the region of helix E (residues 94–102) in the case of CB9 and helix H (residues 148–159) in the case of TH2. In CB9, conformation changes also appear to be transmitted to a portion of the sequence (residues 87–93) preceding helix E, a putative site of interaction between troponin-C and troponin-I. These data are discussed with reference to the contribution of long-range (interdomain) interactions within troponin-C and the modulation of troponin subunit protein-protein interactions by Ca(II) binding.     

Investigation of human erythrocyte superoxide dismutase by 1H nuclear-magnetic-resonance spectroscopy.

The 170MHZ 1 H n.m.r. spectra of the Cu(II)/Zn(II), Cu(I)/Zn(II) and apo- forms of human erythrocyte superoxide dismutase (EC 1.15.1.1) are reported. Resonances are assigned to the C-2 and C-4 protons of histidine residues in the active site, and it is suggested that five or six histidine residues serve as ligands to the metal ions in each subunit of the enzyme. The remaining assigned resonances are associated with histidine-41, N-terminal N-acetyl group, histidine- 108 and cysteine- 109. A comparison of the n.m.r. spectra of human and bovine superoxide dismutases suggests significant structural homology.     

1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.     

Covalent structure of the membranous segment of horse cytochrome b5. Chemical cleavage of the native hemoprotein

The complete covalent structure of the membranous segment of horse liver cytochrome b5 has been determined. This peptide spans residues 91 to 133 of the cytochrome molecule, and contains the segment responsible for the association of the hemoprotein with microsomal or synthetic vesicles. Two peptides, residues 91 to 127 and 128 to 133, comprising the entire membranous moiety were isolated from a tryptic digest of urea-denatured apoprotein. The membranous segment (residues 91 to 127) could be separated from all other tryptic peptides by a single gel filtration step. Trypsin digestion of succinylated cytochrome produced similar peptides, residues 89 to 127 and 128 to 133. The covalent structures for residues 89 to 127 and 128 to 133 were derived from automated sequenator analysis of tryptic peptides. Chemical cleavage at tryptophanyl, or methionyl residues, or both, by the method of Ozols and Gerard ((1977) J. Biol. Chem. 252, 5986-5989) provided the overlapping peptides from which the following unique sequence was deduced: (formula: see text).     

Hydrogen bonding in the carboxyl-terminal half-fragment 78-148 of calmodulin as studied by two-dimensional nuclear magnetic resonance

The C-terminal half-fragment (residues 78-148) of scallop testis calmodulin was investigated by 500-MHz two-dimensional proton NMR in order to clarify the structure and the structural change accompanying Ca2+ binding. The sequential resonance assignment to individual amino acid residues was made in part (27 out of 71 residues) by a combination of correlated spectroscopy and nuclear Overhauser effect spectroscopy of a 90% H2O solution. In the Ca2+-bound state, resonances of backbone amide protons of Gly-98, Gly-134, Ile-100, Asn-137, and Val-136 appear at extremely low fields. These findings suggest that amide protons of these residues are hydrogen bonded. In the Ca2+-free state, the amide resonances of Ile-100 and Gly-134 disappear into the crowded normal shift region. This observation indicates that two hydrogen bonds of Ile-100 and Gly-134 are destroyed (or weakened) as Ca2+ ions are removed from two Ca2+-binding sites. Chemical shifts of amide and alpha-protons of residues located in the Ca2+-binding loop of domain III are similar to those of domain IV. These results suggest that the conformations of the two loops are very similar. The present results can be interpreted in terms of a structure predicted by Kretsinger [Kretsinger, R.H. (1980) Ann. N.Y. Acad. Sci. 356, 14].     

Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin to phosphocholine and ceramide in a divalent metal ion-dependent manner. Bc-SMase is a homologue of mammalian neutral SMase (nSMase) and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, aging, and apoptosis. Thus Bc-SMase may be a good model for the poorly characterized mammalian nSMase. The metal ion activation of sphingomyelinase activity of Bc-SMase was in the order Co2+ > or = Mn2+ > or = Mg2+ > Ca2+ > or = Sr2+. The first crystal structures of Bc-SMase bound to Co2+, Mg2+, or Ca2+ were determined. The water-bridged double divalent metal ions at the center of the cleft in both the Co2+- and Mg2+-bound forms were concluded to be the catalytic architecture required for sphingomyelinase activity. In contrast, the architecture of Ca2+ binding at the site showed only one binding site. A further single metal-binding site exists at one side edge of the cleft. Based on the highly conserved nature of the residues of the binding sites, the crystal structure of Bc-SMase with bound Mg2+ or Co2+ may provide a common structural framework applicable to phosphohydrolases belonging to the DNase I-like folding superfamily. In addition, the structural features and site-directed mutagenesis suggest that the specific beta-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound sphingomyelin substrate.     

19F nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol binding protein II produced in Escherichia coli. An analysis of four tryptophan substitution mutants and their interactions with all-trans-retinol

Rat cellular retinol binding protein (CRBP II) is a 134-amino acid intracellular protein synthesized in the polarized absorptive cells of the intestine. We have previously used 19F nuclear magnetic resonance (NMR) spectroscopy to survey the structural effects of ligand binding on the apoprotein. For these studies, all 4 Trp residues of rat CRBP II were efficiently labeled with 6-fluorotryptophan (6-F-Trp) by inducing its expression in a tryptophan auxotroph of Escherichia coli. Resonances corresponding to 2 of its Trp residues underwent large downfield shifts upon binding of all-trans-retinol and retinal, while resonances corresponding to the other 2 Trp residues underwent only minor perturbations in chemical shifts. To identify which Trp residues undergo changes in their environment upon ligand binding, we have constructed four CRBP II mutants where Trp9, Trp89, Trp107, or Trp110 have been replaced by another hydrophobic amino acid. By comparing the 19F NMR spectrum of each 6-F-Trp-labeled mutant with that of wild type 6-F-Trp CRBP II, we demonstrate that the 19F resonance corresponding to Trp107 undergoes the largest change in chemical shift upon ligand binding (2.0 ppm downfield). This is consistent with the position of this residue predicted from molecular modeling studies. The 19F resonance corresponding to Trp9 also undergoes a downfield change in chemical shift of 0.5 ppm associated with retinol binding even though it is predicted to be removed from the ligand binding site. By contrast, the resonances assigned to Trp89 and Trp110 undergo only minor perturbations in chemical shifts. These results have allowed us to identify residue-specific probes for evaluating the interactions of all-trans-retinol (and other retinoids) with this intracellular binding protein.     

NMR docking of a substrate into the X-ray structure of staphylococcal nuclease.

The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca(2+)-3',5'-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La(3+)-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronuclear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectroscopy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La(3+)-3',5'-pdTp complex and the enzyme-Ca(2+)-3',5'-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5'-dA leaving group to partially overlap the inhibitor, 3',5'-pdTp (in the X-ray structure). The 3'-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5'-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5'-dGMP onto the 3'-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3'-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide.     

Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli. Analysis of the apoprotein and the holoprotein containing bound all-trans-retinol and all-trans-retinal

Rat cellular retinol-binding protein II (CRBP II) is a 15.6-kDa intestinal protein which binds all-trans-retinol and all-trans-retinal but not all-trans-retinoic acid. We have previously analyzed the interaction of Escherichia coli-derived rat apoCRBP II with several retinoids using fluorescence spectroscopic techniques. Interpretation of these experiments is complicated, because the protein has 4 tryptophan residues. To further investigate ligand-protein interactions, we have utilized 19F nuclear magnetic resonance (NMR) spectroscopy of CRBP II labeled at its 4 tryptophan residues with 6-fluorotryptophan. Efficient incorporation of 6-fluorotryptophan (93%) was achieved by growing a tryptophan auxotroph of E. coli harboring a prokaryotic expression vector with a full-length rat CRBP II cDNA on defined medium supplemented with the analog. Comparison of the 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II with and without bound all-trans-retinol revealed that resonances corresponding to 2 tryptophan residues (designated WA and WB) undergo large downfield changes in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand binding. In contrast, 19F resonances corresponding to two other tryptophan residues (WC and WD) undergo only minor perturbations in chemical shifts. The 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II complexed with all-trans-retinal and all-trans-retinol were very similar, suggesting that the interactions of these two ligands with the protein are similar. Molecular model building, based on the crystalline structures of two homologous proteins was used to predict the positions of the 4 tryptophan residues of CRBP II and to make tentative resonance assignments. The fact that ligand binding produced residue-specific changes in the chemical shifts of resonances in CRBP II suggests that NMR analysis of isotopically labeled retinoid-binding proteins expressed in E. coli will provide an alternate, albeit it complementary, approach to fluorescence spectroscopy for examining the structural consequences of their association with ligand.     

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)