Binding of native and [homoserine lactone-52]-52,53-seco-bovine basic pancreatic trypsin inhibitor (kunitz inhibitor) to porcine pancreatic β-kallikrein-B and bovine α-chymtorpsin: Thermodynaic study

Values of the association equilibrium constant (K_a) for the binding of the native and of the cyanogen bromide-cleaved bovine basic pancreatic trypsin inhibitor (native BPTI and [Hse lactone-52]-52,53-seco-BPTI, respectively) to neuraminidase-treated porcine pancreatic β-Kallikrein-B (kallikrein) and bovine α-chymotrypsin (chymotrypsin) have been determined between pH4.0 and 9.0, and 20.0°C. Over the whole pH range explored, native BPTI and [Hse lactone-52]-52,53-seco-BPTI show the same affinity for kallikrein. On the other hand, the affinity of [se lactone-52]-52,53-seco-BPTI for chymotrypsin is high4er, around neutrality, than that found for native BPTI by about one order of magnitude, coverging in the acidic pH limb. The simplest mechanism accounting for the observed data implies that, on lowering the pH from 9.0 to 4.0 (i) the decrease in affinity for the binding of native BPTI to kalikrein and chymotrypsin, as well as for the association of [Hse lactone-52]-52,53-seco-BPTI to kalikrein, reflects the acidic pK shift, upon inhibitor association, of a single inozing group; and (ii) the decrease of K_a values for [Hse lactone-52]-52,53-seco-BPTI binding to chymotrypsin appears to be modulated by the acidic pK shift, upon inhibitor association, of two non-equivalent proton-binding residues. On the basis of the stereochemistry of the serine proteinase/inhibitor contact region(s), these data indicate that long-rang structural changes in [Hse lactone-52]-52,53-seco-BPTI are energetically linked to the chymotrypsin: inhibitor complex formation. This observation represents an important aspect for the mechanism of molecular recognition and regulation in BPTI.     

Peptide models of electrostatic interactions in proteins: NMR studies on two beta-turn tetrapeptides containing Asp-His and Asp-Lys salt bridges

Two model peptides Boc-Asp-Pro-Aib-X-NHMe [X = His (1) and X = Lys (2)] were synthesized to simulate intramolecular electrostatic interactions between ionizable side chains. Conformational analysis by 270-MHz 1H NMR in (CD3)2SO reveals that the backbone secondary structures of these two peptides are stabilized by two strong intramolecular hydrogen bonds, involving the consecutive carboxy-terminal NH groups. 1H NMR chemical shifts were measured in 1, 2, and a protected derivative, Boc-Asp(OBzl)-Pro-Aib-His-NHMe (3). These shifts were also measured for the model compounds Ac-Lys-NHMe, Boc-Asp-NHMe, and Boc-His-NHMe in their different states of ionization. An analysis of the chemical shifts of the ionization-sensitive reporter resonances suggests the formation of a strong intramolecular salt bridge in the lysyl peptide 2 and a bridge of moderate strength in the histidyl peptide 1. A comparison of the temperature dependence of chemical shifts in peptides 1-3 suggests that intramolecular salt bridge formation results in diminished backbone flexibility. The results establish that proximity effects confer far greater stability to intramolecular ion pair interactions vis-a-vis their intermolecular counterparts. The salt bridge interaction in peptide 1 displays a remarkable sensitivity to the dielectric constant of the solvent medium. The results suggest that these peptides are good simulators of the role of salt bridges in the structural dynamics of proteins.     

Sequential assignment of the 1H and 31P resonances of the double stranded deoxynucleotide d (ATGCAT)2 by 2D-NMR correlation spectroscopy

31p-1H and 1H-1H chemical shift correlation spectroscopy are jointly used for providing a complete assignment of sugar proton (except H5' and H5") and phosphorus resonances in the double stranded oligonucleotide d (ATGCAT)2. In contrast to previous methods the specific assignment of overcrowded H5' H5" proton resonances is not required. Using the H3'-P coupling and also the long range H4'-P coupling, this quite general method can be easily implemented on intermediate field spectrometer. The present results pave the way to the 1H and 31P resonance assignment of longer double-stranded oligonucleotides.     

Epidermal growth factor from the mouse. Structural characterization by proton nuclear magnetic resonance and nuclear overhauser experiments at 500 MHz

Mouse epidermal growth factor (mEGF), a protein hormone effector molecule that regulates cellular development and division, has been investigated by using proton nuclear magnetic resonance techniques at 500 MHz. Well-resolved downfield aromatic and alpha-CH proton resonances (5.0-8.0 ppm) and an upfield ring current shifted isoleucine delta-methyl resonance (approximately 0.5 ppm) have been examined by using principally nuclear Overhauser methods. The data are analyzed in terms of model building based on the predictive Chou-Fasman secondary structure algorithm applied to mEGF [Holladay, L. A., Savage, C. R., Cohen, S., & Puett, D. (1976) Biochemistry 15, 2624-2633], which suggests the existence of some beta-structure and little or no alpha-helicity. Proximity relationships derived from nuclear Overhauser data among Tyr-3, -10, and -13, His-22, and Ile-23 allow refinement of some aspects of the predicted secondary structure and render additional information on how the protein backbone in mEGF is folded (i.e., tertiary structure). Nuclear Overhauser effects (NOEs) from irradiation of several alpha-CH proton resonances give evidence for tiered beta-sheet structure in mEGF. Such proximity relationships derived from NOE data place stringent limitations on possible models for the molecule. pH titration data demonstrate a His-22 pKa of 7.1, indicating either a salt bridge or hydrogen-bond formation between His-22 and another residue. The His-22 pKa is also reflected in the chemical shift changes of several other resonances as a function of pH. Nuclear Overhauser methods, used to differentiate direct (protonation) and indirect (conformation) effects on the chemical shift changes in the spectra of mEGF by varying the pH, yield evidence for a pH-induced conformational transition in the protein hormone associated with the breaking of the His-22 salt bridge or hydrogen bond.     

On the pH dependence of amide proton exchange rates in proteins.

We have analyzed the pH dependencies of published amide proton exchange rates (kex) in three proteins: bovine pancreatic trypsin inhibitor (BPTI), bull seminal plasma proteinase inhibitor IIA (BUSI IIA), and calbindin D9K. The base-catalyzed exchange rate constants (kOH) of solvent exposed amides in BPTI are lower for residues with low peptide carbonyl exposure, showing that the environment around the carbonyl oxygen influences kOH. We also examined the possible importance of an exchange mechanism that involves formations of imidic acid intermediates along chains of hydrogen-bonded peptides in the three proteins. By invoking this "relayed imidic acid exchange mechanism," which should be essentially acid-catalyzed, we can explain the surprisingly high pHmin (the pH value at which kex reaches a minimum) found for the non-hydrogen-bonded amide protons in the beta-sheet in BPTI. The successive increase of pHmin along a chain of hydrogen-bonded peptides from the free amide to the free carbonyl, observed in BPTI, can be explained as an increasing contribution of the proposed mechanism in this direction of the chain. For BUSI IIA (pH 4-5) and calbindin D9K (pH 6-7) the majority of amide protons with negative pH dependence of kex are located in chains of hydrogen-bonded peptides; this situation is shown to be consistent with the proposed mechanism.     

Folding of a peptide corresponding to the alpha-helix in bovine pancreatic trypsin inhibitor

A short peptide corresponding to the alpha-helical region of BPTI shows partial folding in aqueous solution (pH 7) as judged by circular dichroism (CD). Folding is temperature and denaturant sensitive, and the peptide is monomeric. The difference CD spectrum, obtained from spectra at two temperatures, indicates that the peptide folds as an alpha-helix. Difference CD spectroscopy provides a sensitive assay for helix formation in peptides exhibiting small amounts of structure. Helix stability in this peptide shows a marked pH dependence which is consistent with stabilizing charged side-chain interactions with the helix dipole and/or salt bridge formation.     

Resonance assignments of non-exchangeable protons in B type DNA oligomers, an overview.

The chemical shifts of 1H resonances of non exchangeable protons (except H5', H5" and adenine H2) of over six hundred nucleotides have been collected. The influence which the base of the nucleotide itself as well as the bases on its 5' and 3' side exert on the chemical shifts of the various resonances has been investigated. Most of the resonances appear to be predominantly influenced by only one base. For H2', H2", H3', H4' and H6/H8 this is the base of the central nucleotide, for H5(C) and CH3(T) it is the one on the 5' side and for H1' it is the one on the 3' side. Chemical shift distribution profiles are presented which allow an estimation of the probability of finding a particular resonance at a particular position in the spectrum.     

Stability studies on derivatives of the bovine pancreatic trypsin inhibitor

Gibbs energy, enthalpy, and entropy data were determined for two selectively modified analogues of bovine pancreatic trypsin inhibitor (BPTI) to provide a model free set of thermodynamic parameters that characterize (a) the energetic and entropic contributions of the 14-38 disulfide bridge and (b) the variation of the overall stability resulting from the introduction of two negative charges into the positions 14 and 38. The two BPTI analogues studied were BPTI having Cys-14 and Cys-38 carboxymethylated (BPTI-RCOM) and BPTI having Cys-14 and Cys-38 carboxamidomethylated (BPTI-RCAM). They were obtained from native BPTI by reduction, followed by modification of the sulfhydryl groups with iodoacetic acid or iodoacetamide, respectively. The temperature dependence of all thermodynamic parameters of BPTI is drastically altered in the absence of the third disulfide bridge. Even the apparently minute difference of two dissociable carboxyl groups instead of uncharged amide groups in positions 14 and 38 has surprisingly large effects on the temperature dependence of the stabilization enthalpy. The Gibbs energy of BPTI at pH 2, 25 degrees C, decreases by approximately 70% when the 14-38 disulfide bond is cleaved. BPTI-RCOM is more stable than BPTI-RCAM in the whole pH range studied. The difference of -4 kJ/mol at pH 2, 25 degrees C, is reduced to -2.7 kJ/mol at pH 5, 25 degrees C. This finding demonstrates that the presence of two negative charges reduces the higher stability of BPTI-RCOM slightly; however, the overall effect of the two charges is still a stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Proton n.m.r. spectroscopic evidence for sulfur-aromatic interactions in peptides

The downfield shift of the tyrosyl proton resonances and an increased chemical shift difference between the resonances for the 2',6' and 3',5' hydrogens in a series of deamino-oxytocin analogs modified in the disulfide bridge provide evidence for aromatic-sulfur interactions in d6-dimethylsulfoxide solutions.     

Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3)

Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu(2+), circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu(2+). The NMR spectra indicate that the addition of small amounts of CuSO(4) to 3 (Y1-G2-H3-G4-G5-G6-Y7) causes significant broadening of resonances of the side chain protons (C(beta)H, C(epsilon1)H, and C(delta2)H) of His3 and the side chain C(beta)H of Tyr1 at pH 7.4. In addition, the backbone C(alpha)H resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu(2+) binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers.     

Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor.

The low-field portions of the 250-MHz 1H nuclear magnetic resonance (NMR) specra of native and chemically modified bovine basic pancreatic trypsin inhibitor (BPTI) have been studied as a function of pH over the range pH 5-13. Resonances associated with the 16 protons of the aromatic rings of the four BPTI tyrosines have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines-10, -21, -23, and -35 of 10.4, 11.0, 11.7, and 11.1, respectively. The resonances associated with the nitrotyrosine-10 protons of mononitrated BPTI and the nitrotyrosine-10 and -21 protons of dinitrated BPTI have been similarly located, assigned and titrated yielding pK's for nitrotyrosine-10 and -21 of 6.6 and 6.4, respectively. The high-field NMR spectrum indicates that the aromatic ring of tyrosine-35 rotates less than 160 times per second at 25 degrees for pH's in the range 5-9.     

Individual assignments of the methyl resonances in the 1H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor.

In earlier work the resonances of the 20 methyl groups in the basic pancreatic trypsin inhibitor (BPTI) had been identified in the 360-MHz 1H nuclear magnetic resonance (NMR) spectra and most of the methyl lines had from spin-decoupling experiments been assigned to the different types of amino acid residues. The assignments to the different amino acid types were now completed by studies of the saturation transfer between the denatured and the globular forms of the inhibitor and by spin-decoupling experiments in nuclear Overhauser enhancement (NOE) difference spectra. These distinguished between the methyl resonances of Ala and Thr. Furthermore, for most of the methyl resonances, individual assignments to specific residues in the amino acid sequence were obtained from measurements of intramolecular proton-proton NOE's, use of lanthanide NMR shift and relaxation probes, and comparative studies of various chemically modified forms of BPTI. These data provide the basis for individual assignments of the methyl 13C NMR lines in BPTI and for detailed investigations of the relations between the spatial structure of the protein and the chemical shifts of the methyl groups. The methyl groups in BPTI are of particular interest since they are located almost exclusively on the surface of the protein and thus represent potential natural NMR probes for studies of the protein-protein interactions in the complexes formed between BPTI and a variety of proteases.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

The amino 1H resonances of oligonucleotide helices: d(CGCG)

An examination of the 1H NMR assignments and exchange properties of the amino resonances of the self-complementary tetramer, d(CGCG) was undertaken with regard to buffer effects, transfer of saturation from the water resonance and temperature dependence of amino 1H line shape and chemical shift. The lack of buffer effect on visible exchangeable proton resonances is evidence for the stringent requirement for nucleo-base protonation at pH values below neutrality, which is greatly reduced in the helical state. For this reason, sharp resonances are observed for both Watson-Crick and non-Watson-Crick cytosine amino protons for base-paired regions. Considerations of monomeric exchange mechanisms for the cytosine and guanine amino protons formed the basis for successful assignment and isolation of their resonances in the helical state by presaturation of the water resonance at selected pH values. Preirradiation of the water resonance at pH less than 6 would isolate the guanine amino 1H resonances of any self-complementary oligonucleotide, to exploit its high sensitivity as a useful proble of helix in equilibrium coil premelting.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides.

The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.     

The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution

Bovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2(1), P6(4)22 and P6(3)22 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers (n=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as "BPTI decamer" crystals.     

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the ¹H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.     

Proton NMR spectroscopy of sulfmyoglobin

The 1H NMR spectra of ferrous sulfmyoglobin, metsulfmyoglobin, and ferric cyanosulfmyoglobin were obtained at 300 MHz. Hyperfine-shifted resonances are observed in the case of metsulfmyoglobin and ferric cyanosulfmyoglobin that have line widths and cover a chemical shift range that are comparable to the corresponding forms of normal myoglobin. Two methyl resonances are observed in the spectrum of ferric cyanosulfmyoglobin at 44.19 and 25.48 ppm (25 degrees C, pH 8.3) that have been assigned to heme methyls at the 8- and 5-positions on the basis of pH titration effects homologous to the corresponding methyl resonances in ferric cyanomyoglobin. Examination of aromatic region resonances and the pH titration profiles of histidine resonances lead to the conclusion that the overall conformation of sulfmyoglobin was highly homologous to that of normal myoglobin and afforded assignments of histidine residues of the former. The most likely position for the addition of a sulfur atom to the heme of sulfmyoglobin is pyrrole ring A, with ring B a possible, but less likely, alternative.