Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change

Rabbit skeletal muscle troponin C (TnC) was investigated by means of 1H NMR in the presence of dithiothreitol that prevents dimerization of the protein. Two-dimensional (2D) 1H NMR spectra were observed in order to assign resonances to specific amino acids. One-dimensional 1H NMR spectra were observed as a function of Ca2+ concentration. The Ca2+-induced spectral change is categorized into two types: type 1 corresponds to the conformational change of the C-terminal-half domain (Ca2+ high-affinity sites) and type 2 to that of the N-terminal-half domain (Ca2+ low-affinity sites). From the 2D NMR spectra and Ca2+ titration data, it was suggested that (1) amide protons of Gly-108, Ile-110, Gly-144, and Ile-146 are hydrogen-bonded when the C-terminal-half domain binds 2 mol of Ca2+ and (2) hydrogen bonds of Gly-108, Ile-110, Gly-144, and Ile-146 are destroyed or weakened when the C-terminal-half domain releases 2 mol of Ca2+. Nuclear Overhauser enhancement difference spectra as well as the Ca2+ titration data suggested that a hydrophobic cluster is formed in the C-terminal-half domain when the C-terminal-half domain binds 2 mol of Ca2+. A hydrophobic cluster exists in the N-terminal-half domain without regard to Ca2+ binding to the N-terminal-half domain. The spectra of Tyr-10 showed both types of spectral change during the Ca2+ titration. The results suggested that Tyr-10 of apo-TnC interacts with the C-terminal-half domain.     

1H NMR studies of mastoparan binding by calmodulin

Association with the cytoactive tetradecapeptide mastoparan perturbs the downfield 1H NMR spectrum of the calmodulin-Ca42+ complex. Changes occur in the resonances assigned to His-107 and Tyr-138. Composite peaks assigned to Phe-16 and Phe-89 and to Phe-68 and Tyr-99 are also affected. Both the upfield and downfield 1H NMR spectra contain evidence for spectroscopically distinct intermediates in Ca2+ binding by the calmodulin-mastoparan complex.     

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].     

Application of peptide-mediated ring current shifts to the study of neurophysin-peptide interactions: a partial model of the neurophysin-peptide complex

Perdeuteriated peptides were synthesized that are capable of binding to the hormone binding site of neurophysin but that differ in the position of aromatic residues. The binding of these peptides to bovine neurophysin I and its des-1-8 derivative was studied by proton nuclear magnetic resonance spectroscopy in order to identify protein residues near the binding site through the observation of differential ring current effects on assignable protein resonances. Phenylalanine in position 3 of bound peptides was shown to induce significant ring current shifts in several resonances assignable to the 1-8 sequence, including those of Leu-3 and/or Leu-5, but was without effect on Tyr-49 ring protons. The magnitude of these shifts was dependent on the identity of peptide residue 1. By contrast, the sole demonstrable direct effect of an aromatic residue in position 1 was a downfield shift in Tyr-49 ring protons. Study of peptide binding to des-1-8-neurophysin demonstrated similar conformations of native and des-1-8 complexes except for the environment of Tyr-49, confirmed the peptide-induced ring current shift assignments in native neurophysin, and indicated an effect of binding on Thr-9. These observations are integrated with other results to provide a partial model of neurophysin-peptide complexes that places the ring of Tyr-49 at a distance 5-10 A from residue 1 of bound peptide and that places both the 1-8 sequence and the protein backbone region containing Tyr-49 proximal to each other and to peptide residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.     

Assignment of asparagine-44 side-chain primary amide 1H NMR resonances and the peptide amide N1H resonance of glycine-37 in basic pancreatic trypsin inhibitor

New assignments of three previously undetected amide proton NMR resonance lines in bovine pancreatic trypsin inhibitor are reported. These are the peptide amide proton of Gly-37 and the primary amide protons of Asn-44. Specific assignments of Asn-44 and Asn-43 HE and HZ resonances are also reported. The Gly-37 NH and Asn-44 HZ resonances are shifted upfield to 4.3 and 3.4 ppm, respectively, by the ring current of the Tyr-35 aromatic group, while Asn-44 HE resonates at 7.8 ppm. The abnormal chemical shifts of Asn-44 HZ and Gly-37 NH indicate that both NH's interact with the pi-electron cloud of the Tyr-35 ring. This is consistent with their location in the crystal structure. The resonances are resolved by differential labeling techniques and are studied by combined use of NOE and exchange difference spectroscopy.     

Sulfation of gastrin: Effect on immunoreactivity

The effect of sulfuric acid esterification of Tyr-12 in gastrin-17 on immunoreactivity was evaluated by the ability of seventeen antisera raised against non-sulfated gastrin-17 to bind sulfated gastrins in extracts of gastrinoma and antral tissue. Using non-sulfated Tyr-12 iodinated gastrin as tracer, and non-sulfated gastrin-17 as standard the antisera showed three different patterns of reactivity: Three antisera (Nos. 2602, 2605 and 4562) bound sulfated gastrins with low (4–23%) potency; four antisera (Nos. 2604, 2720, 4710 and 4713) measured sulfated gastrins with a potency similar to that of non-sulfated gastrins (81–100% crossreactivity); whereas ten antisera (Nos. 2601, 2606, 2609, 2716, 2717, 2718, 4556, 4559, 4560 and 4563) displayed enhanced reactivity with sulfated gastrins (130–373% cross-reactivity). Using Gly-2 iodinated gastrin as tracer, the latter type of antisera reacted almost equally with sulfated and non-sulfated gastrins, suggesting that the apparent increase in binding of sulfated gastrins rather is due to increased displacement of Tyr-12 iodinated gastrin. The results show that derivatization of amino acid residues greatly influences antibody binding.     

Domain interactions in human plasminogen studied by proton NMR.

The NMR spectrum of miniplasminogen (V443-plasminogen) under conditions of acidic pH reveals a subset of particularly well-resolved resonances whose chemical shift values are closely similar to those of isolated kringle 5. The temperature dependence of the spectrum indicates that this set of resonances disappears in a single cooperative unfolding transition appropriate for kringle 5, whilst other broader resonances from the protease domain persist to higher temperature. These results provide evidence for significant structural and motional independence of the kringle and protease domains in spite of the short linker between these domains. The NMR spectrum of Glu1-plasminogen is closely similar to that of miniplasminogen under the same conditions. This suggests that the domain independence observed in miniplasminogen is maintained in the intact molecule.     

Human platelet factor 4 monomer-dimer-tetramer equilibria investigated by 1H NMR spectroscopy

As a function of protein concentration, proton NMR spectra of human platelet factor 4 (PF4) differ. Correlation with low-angle laser light scattering data has allowed identification of concentration-dependent NMR spectral changes to PF4 aggregation, with tetramers being the largest aggregates formed. Well-resolved aromatic ring proton NMR resonances were assigned to Tyr-60, His-I, and His-II in monomer, dimer, and tetramer states. Since Tyr-60 3.5 ring proton resonances are well resolved from state to state, estimation of fractional populations in each state was possible. By varying the PF4 concentration, changes in these populations when plotted according to the Hill equation show a bimolecular mechanism of aggregation which proceeds from monomers to tetramers through a dimer intermediate. Equilibrium constants for dimer association (KD) and tetramer association (KT) have been estimated as a function of pH and ionic strength. At pH 4, where KD and KT approach the same value, resonances associated with all three aggregate states are observed. Lowering the pH shifts the equilibrium to the monomer state, while raising the pH shifts the equilibrium to dimer and tetramer states. Analysis of the pH dependence of KD and KT suggests that electrostatic interactions, probably arising from Glu/Asp and Lys/Arg side chains, play a role in the binding process. Increasing the solvent ionic strength stabilizes the tetramer state especially at low pH, suggesting that intersubunit, repulsive electrostatic interactions probably between/among cationic side chains (Lys/Arg) attenuate the aggregation process. Information based primarily on histidine pKa values and photo-CIDNP 1H NMR data suggests that Tyr-60 and His-I, but not His-II, are significantly affected by the aggregation process.     

Use of perdeuterated peptides in NMR studies of neurophysin-hormone interaction: demonstration of peptide-specific changes in neurophysin resonances

The effects on bovine neurophysin-I of binding the perdeuterated peptides Phe-PheNH2 and Leu-PheNH2 were compared by proton NMR. A unique difference between the two peptides in their effects on Tyr-49 ring protons indicated proximity of the Tyr-49 ring to the side-chain of position 1 of bound peptide. Non-deuterated oligopeptides containing Phe in position 3 and no methyl groups induced different changes in neurophysin methyl resonances than dipeptides, suggesting shielding of one or more protein methyl groups by Phe-3. The results demonstrate that the identity of neurophysin residues at the hormone-binding site can be probed by analysis of changes induced in the protein spectrum by systematically related NMR-transparent peptides.     

NMR study of the phosphoryl binding loop in purine nucleotide proteins: evidence for strong hydrogen bonding in human N-ras p21

The structure of the phosphoryl binding region of human N-ras p21 was probed by using heteronuclear proton-observed NMR methods. Normal protein and a Gly-12----Asp-12 mutant protein were prepared with two amino acids labeled with 15N at their amide positions: valine and glycine, aspartic acid and glycine, and lysine and glycine. We completed the identification of amide 15NH resonances from Gly-12 and Asp-12 to the end of the phosphoryl binding domain consensus sequence (Lys-16) in protein complexed with GDP and have made tentative amide identifications from Val-9 to Ser-17. The methods used, together with initial identifications of the Gly-12 and -13 amide resonances, were described previously [Campbell-Burk, S. (1989) Biochemistry 28, 9478-9484]. The amide resonances of both Gly-13 and Lys-16 are shifted downfield below 10.4 ppm in both the normal and mutant proteins. These downfield shifts are presumed to be due to strong hydrogen bonds with the beta-phosphate oxygens of GDP.     

Calcium binding to the isolated beta-hydroxyaspartic acid-containing epidermal growth factor-like domain of bovine factor X

Coagulation factor X is a vitamin K-dependent protein composed of discrete domains or modules. A proteolytically modified derivative of factor X that lacks the NH2-terminal gamma-carboxyglutamic acid (Gla)-containing region retains one Ca2+ binding site. To localize this Gla-independent Ca2+ binding site and to facilitate future studies aimed at elucidating structure-function relationship in the factor X molecule, we have devised a method to isolate the first beta-hydroxyaspartic acid (Hya)-containing epidermal growth factor (EGF)-like domain from proteolytic digests of bovine factor X performed under strictly controlled conditions. The EGF-like domain, corresponding to residues 45-86 in bovine factor X, was obtained in more than 50% recovery, and was at least 98% homogeneous as judged by NH2-terminal sequence analysis. Ca2+ binding to the isolated EGF-like domain was studied by 1H NMR spectroscopy. On binding of Ca2+ to the domain the resonances from Tyr-68 centered at 6.8 ppm were affected. The Ca2+ concentration dependence of the chemical shift was used to calculate the Ca2+ binding constant, resulting in a K alpha of 4 X 10(3) M-1 at pH 8.5 and 1 X 10(3) M-1 at pH 7.4, the higher value presumably reflecting an increase in negative surface charge due to deprotonation of a histidine residue with a pK alpha of 7.4. The NMR spectra gave no evidence of a conformational change in the EGF-like domain between pH 6 and 8.5.     

Nuclear-magnetic-resonance studies of ferrocytochrome c. pH and temperature dependence

The pH dependence and the temperature dependence of the nuclear magnetic resonance spectrum of horse ferrocytochrome c are described. This protein is very stable; it maintains an ordered structure over the pH range 4 to 12 at 25 degrees C and over the temperature range 4 degrees C to 97 degrees C at pH 7.0. The dynamic characteristics of the conformation of ferrocytochrome c were investigated. Particular emphasis was laid on the aromatic resonances and resonances of methyl groups shifted far upfield. Tyr-48 and Phe-46 were found to be relatively immobile whilst a region of the protein close to Ile-57 was found to be relatively flexible.     

Slowly interchanging conformers of bovine neurophysin-I in the unliganded dimeric state.

The effect of neurophysin dimerization on Tyr-49, a residue adjacent to the hormone-binding site, was investigated by proton NMR in order to analyze the basis of the dimerization-induced increase in neurophysin hormone affinity. Dimerization-induced changes in Tyr-49 resonances, in two unliganded bovine neurophysins, suggested that Tyr-49 perturbation is an intrinsic consequence of dimerization, although Tyr-49 is distant from the monomer-monomer interface in the crystalline liganded state. To determine whether this perturbation reflects a conformational difference between liganded and unliganded states that places Tyr-49 at the interface in the unliganded state, or a dimerization-induced change in secondary (2 degrees) or tertiary (3 degrees) structure, the more general structural consequences of dimerization were further analyzed. No change in 2 degrees structure upon dimerization was demonstrable by CD. On the other hand, a general similarity of regions involved in dimerization in unliganded and liganded states was indicated by NMR evidence of participation of His-80 and Phe-35 in dimerization in the unliganded state; both residues are at the interface in the crystal structure and distant from Tyr-49. Consistent with a lack of direct participation of Tyr-49 at the monomer-monomer interface, dimerization induced at least two distinct slowly exchanging environmental states for the 3.5 ring protons of Tyr-49 without significantly increased dipolar broadening relative to the monomer. Two environments were also found in the dimer of des-1-8 neurophysin-I for the methyl protons of Thr-9, another residue distant from the monomer-monomer interface and close to the binding site in the liganded state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). (13)C and (15)N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding, and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, nonideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and nonideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (?, ψ) torsion angles for three "basis" states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding, and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins.     

Conformational heterogeneity in polypeptide cardiac stimulants from sea anemones

High-resolution 1H NMR spectra at 300 MHz of the polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxin II reveal conformational heterogeneity in both molecules. The two conformations, manifest in a number of split 1H resonances, are in slow exchange over a wide range of pH and temperature. Heterogeneity affects a region of these molecules containing the structurally and functionally important Asp residues. By comparison with a homologous polypeptide Anemonia sulcata toxin I, which does not show this type of heterogeneity, it is suggested that the heterogeneity may originate in cis-trans isomerism of the Gly-40 to Pro-41 peptide bond.     

Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase

The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin.

Two series of site-directed mutations to the individual Ca(2+)-binding sites of Drosophila melanogaster calmodulin have been generated and studied. In each mutant, a conserved glutamic acid residue at position 12 in all of the Ca(2+)-binding loops has been mutated in one site. In one series the residue is changed to glutamine; in the second series the change is to lysine. The Ca(2+)-binding properties of these mutants and the wild-type protein under pseudo-physiological conditions are presented. In addition, Ca(2+)-induced changes to the environment of the single tyrosine residue (Tyr-138) have been studied for some of the mutants. Ca2+ binding to the wild-type protein is best modeled as two pairs of sites with a higher affinity pair that shows strong cooperativity. For all but one of these eight mutant proteins, only three Ca(2+)-binding events can be detected. In three of the amino-terminal mutants, the three residual sites are (i) a pair of relatively high affinity sites and (ii) a weakened low affinity site. For all four carboxyl-terminal mutations, the residual sites are three relatively low affinity sites. In general, mutations to sites 2 and 4 prove more deleterious than mutations to sites 1 and 3. The Ca(2+)-induced conformational changes in the vicinity of Tyr-138 are relatively undisturbed by mutations of site 1. However, the changes to Tyr-138 in the carboxyl-terminal site mutants indicate that upon disruption of the cooperative binding at the high affinity sites, conformational change in the carboxyl terminus occurs in two phases. It appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site. The final conformations of site 3 and 4 mutants are thus significantly different.     

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)     

Proton magnetic resonance and binding studies of proteolytically modified neurophysins

The proton NMR spectra and role in peptide binding of carboxyl-terminal and NH2-terminal neurophysin residues were studied by preparation of bovine neurophysin-I derivatives from which residues 90-92 had been cleaved by carboxypeptidase or residues 1-8 excised by trypsin. The carboxypeptidase-treated protein showed normal peptide-binding behavior. NMR comparisons of this derivative and the native protein allowed identification of proton resonances associated with residues 89-92, confirmed a lack of functional role for this region of the protein, and permitted new observations on the behavior of neurophysin's aromatic residues. The trypsin-treated protein bound peptide with an affinity only 1/50 that of the native protein at pH 6 but evinced the same binding specificity and pH dependence of binding as the native protein. These results argued against direct interaction of residues in the 1-8 sequence with bound peptide and for a role for these residues, particularly Arg-8, in conformational stabilization of the active site; this role is held to be additional to the reported influence of 1-8 on dimerization. NMR comparisons of the trypsin product and native protein allowed preliminary assignment of a set of alkyl proton resonances to residues within the 1-8 sequence and were compatible with a restricted environment for Arg-8. Conformational differences between native and trypsin-treated proteins were manifest particularly by differences in the NMR spectra of Phe and Tyr-49 ring protons. The behavior of Phe ring protons was consistent with the reported decreased dimerization constant of the trypsin product and suggested participation of Phe-22 or -35 in dimerization. The behavior of Tyr-49 provided the first direct evidence of a change in secondary or tertiary structure associated with excision of residues 1-8. Suggested mechanisms by which this conformational change reduces binding include a direct effect on Tyr-49 and/or a conformational rearrangement of active site residues near Tyr-49.