Intestinal fatty acid binding protein: the folding mechanism as determined by NMR studies

The intestinal fatty acid binding protein is composed of two beta-sheets surrounding a large interior cavity. There is a small helical domain associated with the portal for entry of the ligand into the cavity. Denaturation of the protein has been monitored in a residue-specific manner by collecting a series of two-dimensional (1)H-(15)N heteronuclear single-quantum coherence (HSQC) NMR spectra from 0 to 6.5 M urea under equilibrium conditions. In addition, rates for hydrogen-deuterium exchange have been measured as a function of denaturant concentration. Residual, native-like structure persists around hydrophobic clusters at very high urea concentrations. This residual structure (reflecting only about 2-7% persistence of native-like structure) involves the turns between beta-strands and between the two short helices. If this persistence is assumed to reflect transient native-like structure in these regions of the polypeptide chain, these sites may serve as nucleation sites for folding. The data obtained at different urea concentrations are then analyzed on the basis of peak intensities relative to the intensities in the absence of urea reflecting the extent of secondary structure formation. At urea concentrations somewhat below 6.5 M, specific hydrophobic residues in the C-terminal beta-sheet interact and two strands, the D and E strands in the N-terminal beta-sheet, are stabilized. These latter strands surround one of the turns showing residual structure. With decreasing urea concentrations, the remaining strands are stabilized in a specific order. The early strand stabilization appears to trigger the formation of the remainder of the C-terminal beta-sheet. At low urea concentrations, hydrogen bonds are formed. A pathway is proposed on the basis of the data describing the early, intermediate, and late folding steps for this almost all beta-sheet protein. The data also show that there are regions of the protein which appear to act in a concerted manner at intermediate steps in refolding.     

Inactivation of B12 and folate coenzymes by butyl nitrite as observed by NMR: implications on one-carbon transfer mechanism

The effects of butyl nitrite, a frequently used recreational drug, on methyl cobalamin and 5-methyl tetrahydrofolate were investigated by using 1H and 31P NMR spectroscopies. While no effect could be observed in organic solvents, strong interactions of butyl nitrite with the methyl cobalamin and 5-methyl tetrahydrofolate were found to occur in water. Butyl nitrite decomposes in water generating H+ and NO-2. The former protonates to give the "base-off" configuration of methyl cobalamin while the Co-CH3 bond is cleaved. Similarly, the methyl group at the 5N position and the pyrazine ring of 5-methyl tetrahydrofolate were found to be affected by butyl nitrite. The overall interaction of butyl nitrite with both coenzymes shows displacement of the methyl group and derivatization or destruction of the coenzymes that may lead to deficiencies of both B-12 and/or folates.     

Amide proton exchange rates of a bound pepsin inhibitor determined by isotope-edited proton NMR experiments

From a series of isotope-edited proton NMR spectra, amide proton exchange rates were measured at 20 degrees C, 30 degrees C, and 40 degrees C for a tightly bound 15N-labeled tripeptide inhibitor of porcine pepsin (IC50 = 1.7 X 10(-) M). Markedly different NH exchange rates were observed for the three amide protons of the bound inhibitor. The P1 NH exchanged much more slowly than the P2 NH and P3 NH. These results are discussed in terms of the relative solvent accessibility in the active site and the role of the NH protons of the inhibitor for hydrogen bonding to the enzyme. In this study a useful approach is demonstrated for obtaining NH exchange rates on ligands bound to biomacromolecules, the knowledge of which could be of potential utility in the design of therapeutically useful nonpeptide enzyme inhibitors from peptide leads.     

Metabolism-independent binding of toxic metals by Ulva lactuca: cadmium binds to oxygen-containing groups, as determined by NMR

The metabolism-independent metal binding characteristics of Ulva lactuca were investigated using both freeze-dried thalli and cell walls stripped of intracellular material by incubation in Triton-X followed by methanol. Biosorption of Cd, Zn, Cu and Co by freeze-dried thallus was concentration-dependent and followed Freundlich and Langmuir isotherms. The Freundlich plot suggested that freeze-dried U. lactuca had the greatest binding affinity for Cu compared with Cd, Zn and Co. The BET (Brunauer–Emmett–Teller) plot, which indicates a more complex form of adsorption, and the Scatchard plot were not adequate models for Cu adsorption. The Scatchard plot of Cd suggested that two Cd binding sites were available on the freeze-dried thallus, with the second, lower affinity site only becoming available at Cd loading capacities greater than 4.9mmol g dry wt. Cd nuclear magnetic resonance (NMR) studies confirmed that two binding sites were available for Cd on the freeze-dried algal powder, though only one was available on the cell wall, and that the affinity of the binding sites was greater for Cu than for Cd. The results of the NMR experiments suggested that Cd binds to oxygen-containing functional groups in the algal powder and on the cell wall. It is proposed that sulphate or hydroxyl groups attached to polysaccharide subunits are possible sites.     

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.     

Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR.

Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.     

Electron redistribution on binding of a substrate to an enzyme: folate and dihydrofolate reductase

The migration of electron density of a substrate (folate) on binding to an enzyme (dihydrofolate reductase) is studied by a quantum-mechanical method originally developed in solid state physics. A significant polarization of the substrate is induced by the enzyme, toward the transition state of the enzymatic reaction, at the same time giving rise to "electronic strain energy" in the substrate and enhanced protein-ligand interactions. The spatial arrangement of protein charges that induces the polarization is identified and found to be structurally conserved for bacterial and vertebrate dihydrofolate reductases.     

Type I collagen alpha-1 chain C-telopeptide: solution structure determined by 600-MHz proton NMR spectroscopy and implications for its role in collagen fibrillogenesis

The solution conformation of the alpha-1 chain C-telopeptide has been studied by circular dichroism (CD) and 600-MHz 1H NMR spectroscopy in 60% CD3OH/40% H2O solution. The C-telopeptide contains 27 amino acids which form the C-terminal end of the alpha-1 collagen polypeptide chain. By the combined application of various two-dimensional, phase-sensitive NMR techniques (COSY, RELAY, NOESY, ROESY), a nearly complete assignment of all proton resonances was achieved. Furthermore, the backbone conformation could be established, on the basis of coupling constant and NOE data. The spectroscopic evidence indicates that large sections of the peptide exist in a nonrandom, extended conformation and that there are two segments of higher mobility around the two Gly-Gly units in positions 2,3 and 20,21. Despite these hingelike, flexible sections no measurable fold-back of any of the extended parts was evident. On the basis of this structure, a model is proposed for the simultaneous interaction of the C-telopeptide with two adjacent collagen triple helices within the growing collagen fibril.     

Sterol orientations in phosphatidylcholine liposomes as determined by deuterium NMR

Deuterium magnetic resonance spectra (55.26 MHz) of cholesterol-3 alpha-d1 and epicholesterol-3 beta-d1 in dipalmitoylglycerophosphocholine (DPPC) liposomes were measured as a function of sterol-to-phospholipid ratio below (24 degrees C) and above (60 degrees C) the phase transition temperature of DPPC. From the quadrupolar splittings delta vq, the molecular order parameters S describing the motions of the sterols in the bilayer were calculated, and the most probable angle of tilt alpha 0 of the molecular axis of the sterols relative to the bilayer normal was determined. We observed that the molecular axis of cholesterol in DPPC liposomes at both 24 and 60 degrees C is tilted at an angle of 16-19 degrees with the 3 beta-hydroxyl group projecting parallel to the bilayer normal into the aqueous interface. In contrast, at 24 degrees C, epicholesterol is aligned parallel (0 degrees) to the bilayer normal, placing the 3 alpha-hydroxyl group essentially perpendicular to the bilayer normal along the aqueous interface. At 60 degrees C, the average angle of epicholesterol (16-18 degrees) is similar to that of cholesterol, which can project the 3 alpha-hydroxyl group into the hydrophobic bilayer region. On the basis of the observed tilt angles of the two isomeric sterols in DPPC liposomes, a model is proposed that can rationalize the differential effects of cholesterol and epicholesterol on membrane properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

Conformation of the ATP binding peptide in actin revealed by proton NMR spectroscopy

The actin peptide 106-124 exists in a completely conserved region of the sequence and binds strongly to both ATP and tripolyphosphate. Binding particularly affects residues 116 and 118 and generally affects the two segments 115-118 and 121-124 [Barden, J. A., & Kemp, B. E. (1987) Biochemistry 26, 1471-1478]. One-dimensional nuclear Overhauser enhancement difference spectroscopy was used to detect molecular interactions between both adjacent and nonadjacent residues. The N-terminal segment 106-112 was found to be largely extended. A sharp bend was detected between Pro-112 and Lys-113. The triphosphate moiety binds to the strongly hydrophilic central segment of the peptide. Evidence was obtained for a reverse turn involving residues 121-124. Amide proton temperature coefficients and coupling constants provide evidence for a type I beta-turn. A model of the ATP binding site is proposed together with its relationship to other parts of the actin structure and to the phalloidin binding site.     

Abasic frameshift in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations

We have determined the three-dimensional structure of a non-self-complementary oligodeoxynucleotide duplex that contains a model abasic site. The duplex contains six GC base pairs plus the abasic site at the center of one strand and corresponds to an abasic frameshift. Two-dimensional NMR studies on the nonexchangeable protons show that the guanine bases on either side of the abasic site are stacked over each other and that the abasic site is rotated out of the helix. Close proton-proton interactions are observed between the H4' proton of the abasic site and sugar protons of the guanosine in the 5' direction, which allows the position of the free sugar to be well-defined. NOE buildup curves from NOESY spectra recorded at very short mixing times were used to calculate a set of interproton distances. This data set was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two conformations that differ in the sugar conformation of the guanosine next to the abasic site in the 3' direction were necessary to fit all the NMR data. One of these two conformations could only be stabilized by addition of counterions at specific sites.     

Analysis of proton release in oxygen binding by hemoglobin: implications for the cooperative mechanism

The relationship in hemoglobin between cooperativity (dependence of the Hill constant on pH0 and the Bohr effect (dependence of the mean oxygen affinity on pH) can be described by a statistical thermodynamic model [Szabo, A., & Karplus, M. (1972) J. Mol. Biol. 72, 163-197; Lee, A., & Karplus, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7055-759]. In this model, salt bridges and other interactions serve to couple tertiary and quaternary structural changes. To test and refine the model, it is applied to the analysis of the pH dependence of the tetramer Adair constants corrected for statistical factors (K4i', i = 1-4). Attention is focused on the proton release of the first (delta H1+ = alpha log K41'/alpha pH) and last (delta H4+ = alpha log K44'/alpha pH) oxygenation steps, where K4i' are the Adair constants corrected for statistical factors. Measurements of delta H1+ and delta H4+ under carefully controlled conditions are reported, and good agreement between the model calculation and these experimental results is obtained. The salt bridges are found to be partially coupled to the ligation state in the deoxy quaternary structure; it is shown that a Monod-Wyman-Changeux-type model, in which the salt bridges are coupled only to quaternary structural change, is inconsistent with the data for delta H1. The significance of the present analysis for an evaluation of the Perutz mechanism [Perutz, M.F. (1970) Nature (London) 228, 726-734, 734-739] and other models for hemoglobin cooperativity is discussed.     

Orientational order of Australian spider silks as determined by solid-state NMR

A simple solid-state NMR method was used to study the structure of (13)C- and (15)N-enriched silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. Carbon-13 and (15)N spectra from alanine- or glycine-labeled oriented dragline silks were acquired with the fiber axis aligned parallel or perpendicular to the magnetic field. The fraction of oriented component was determined from each amino acid, alanine and glycine, using each nucleus independently, and attributed to the ordered crystalline domains in the silk. The relative fraction of ordered alanine was found to be higher than the fraction of ordered glycine, akin to the observation of alanine-rich domains in silk-worm (Bombyx mori) silk. A higher degree of crystallinity was observed in the dragline silk of N. edulis compared with A. keyserlingi, which correlates with the superior mechanical properties of the former.     

Metal binding to angiotensin converting enzyme: implications for the metal binding site

Angiotensin converting enzyme interacts with the chelator, 1,10-phenanthroline (OP) to form an OP-Zn-ACE ternary complex, which subsequently dissociates to OP-Zn and apoenzyme. The association and dissociation rate constants for the reaction OP + Zn-ACE in equilibrium OP-Zn-ACE have been determined and compared with those of known OP-metal complexes. Such constants were also used to calculate the rate constant for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissociation of zinc from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concentration, at different pH values, and with different buffers. The stability constant for the binding of zinc to apoACE log Kc = 8.2, determined by equilibrium dialysis using atomic absorption spectroscopy to assess metal concentration, is much smaller than that for Zn-carboxypeptidase A. Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the zinc dissociation rate constant of ACE, 7.5 X 10(-3) sec-1 at pH 7.0, which is much greater than that of the other zinc metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.     

An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations

We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.     

Peptidyl-prolyl cis-trans isomerase activity as studied by dynamic proton NMR spectroscopy

Recently the identity of the peptidyl-prolyl cis-trans isomerase (PPIase), which accelerates the cis/trans isomerization of prolyl peptide bonds and cyclophilin, the binding protein for the immunosuppressive drug Cyclosporin A (CsA), was discovered. The PPIase catalysis toward the substrate Suc-Ala-Phe-Pro-Phe-pNA has been studied by 1H NMR spectroscopy. Using the bandshape analysis technique the rate of interconversion between the cis and trans isomers of the substrate could be measured in the presence of PPIase and under equilibrium conditions. The acceleration is inhibited by equimolar amounts of CsA. The results provide evidence that the PPIase catalysis is more complex than a simple exchange between two states.     

Structure and dynamics of hydrated statherin on hydroxyapatite as determined by solid-state NMR.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicate the N-terminus is alpha-helical in character with the residues directly binding to the HAP being stabilized in the alpha-helical conformation by the presence of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of statherin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain outside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.