Nature of lysozyme-water interactions by proton NMR.

Proton spin-lattice relaxation measurements were performed in 10 mM lysozyme solution as a function of temperature and degree of substitution of solvent H2O with D2O. The results show that in the temperature range from 274 to 323 K, the intermolecular lysozyme proton water proton coupling contributes appreciably to the observed water proton relaxation rate. In this system exchange between water protons and labile protein protons does not dominate the behaviour with temperature of the water-lysozyme intermolecular contribution to the spin-lattice relaxation.     

Domain structure and interactions of recombinant urokinase-type plasminogen activator.

Urokinase-type plasminogen activator (uPA) is a mosaic glycoprotein composed of an epidermal growth factor-like (EGF), a kringle and a serine protease (SP) module. It exists in single and two-chain forms designated HMW pro-uPA and HMW uPA, respectively. A low molecular weight form, LMW uPA, lacks the EGF and kringle modules and is composed of the SP module alone. Recombinant-expressed proteins representing both HMW forms exhibit four reversible unfolding transitions that are resolved by deconvolution of melting curves obtained by differential scanning calorimetry at pH 4.5; no differences in the melting properties of the single and two-chain forms were found. The proteolytic fragment Ser1-Lys135 (EGF-kringle) exhibits two transitions, while the isolated EGF and kringle modules each exhibit a single two-state transition. Thus, both of these modules retain an independently folded compact structure when isolated. The isolated SP module (LMW uPA) exhibits two closely spaced transitions at low pH indicating the melting of two domains of similar stability. Fluorescence-detected melting curves of LMW uPA reveal increasing cooperativity with increasing pH, suggesting an increase in the interaction between the two SP domains. Treatment of both HMW and LMW uPA with the tripeptide inhibitor Glu-Gly-Arg-chloromethylketone dramatically increased the stability of both domains of the SP module which now melt together in a single two-state transition, even at low pH, with no effect on the EGF and kringle modules. From these data one concludes that UK consists of four independently folded domains. Two are formed by the EGF and kringle modules which do not interact with each other or with the SP module. The SP module contains two domains that are independent at low pH but exhibit a tendency to merge into a single cooperative unit at neutral pH or after treatment with the tripeptide inhibitor.     

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.     

Water exchange in plant tissue studied by proton NMR in the presence of paramagnetic centers.

The proton NMR relaxation of water in maize roots in the presence of paramagnetic centers, Mn2+, Mn- EDTA2 -, and dextran-magnetite was measured. It was shown that the NMR method of Conlon and Outhred (1972, Biochem. Biophys. Acta. 288:354-361) can be applied to a heterogenous multicellular system, and the water exchange time between cortical cells and the extracellular space can be calculated. The water exchange is presumably controlled by the intracellular unstirred layers. The Mn- EDTA2 - complex is a suitable paramagnetic compound for complex tissue, while the application of dextran-magnetite is probably restricted to studies of water exchange in cell suspensions. The water free space of the root and viscosity of the cells cytoplasm was estimated with the use of Mn- EDTA2 -. The convenience of proton NMR for studying the multiphase uptake of paramagnetic ions by plant root as well as their transport to leaves is demonstrated. A simple and rapid NMR technique (spin-echo recovery) for continuous measurement of the uptake process is presented.     

Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR

Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used ³¹P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and ³¹P T₂ relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T₂ relaxation are observed at higher temperatures. A comparison of ³¹P T₁ values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T₁ relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules  – in particular for the negatively charged DMPG – while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide–membrane interactions.     

Domain structure and domain-domain interactions of recombinant tissue plasminogen activator

The melting of recombinant tissue plasminogen activator (rtPA) has been investigated by differential scanning calorimetry and fluorescence spectroscopy. At neutral pH, rtPA melts with only partial reversibility in a single sharp peak that can be deconvoluted into four transitions. By contrast, at acidic pH the melting process is spread over a broad range of temperature and is highly reversible. Under these conditions five transitions are resolved by deconvolution analysis. Additional measurements in 6 M guanidinium chloride reveal a sixth transition representing an extremely stable domain. Comparison of the melting curves of several fragments with those of the parent protein allowed all of the transitions to be assigned. The results indicate that rtPA is comprised of six independently folded domains. Two of these domains correspond to the two kringle modules whose thermodynamic properties are similar to those of the kringles in plasminogen. Two additional domains are formed by the epidermal growth factor (EGF)-like and finger modules, the latter of which is extremely stable, requiring the presence of a chemical denaturant for its melting to be observed. The serine protease module contains two more domains which at neutral pH melt cooperatively in a single transition but at low pH melt independently, accounting for the greater number of transitions observed there. Measurements with a 50-kDa fragment lacking the C-terminal half of the serine protease module and with a variant lacking the finger and EGF domains indicate that the serine protease domains interact strongly with and are stabilized by the finger and/or EGF domains in the intact protein. This interaction between domains located at opposite ends of the rtPA molecule produces a more compact structure. A better understanding of such interactions may enhance efforts to engineer plasminogen activators with improved thrombolytic properties.     

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.     

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.     

Ligand interactions with the kringle 5 domain of plasminogen. A study by 1H NMR spectroscopy

The binding of small molecules to the kringle 5 domain fragment of human plasminogen has been investigated by 1H NMR spectroscopy at 300 MHz. The compounds tested as potential ligands include L-arginine, L-lysine, and a number of aliphatic and aromatic analogs of similar size but different ionic charge configurations. Ligand/kringle 5 association constant (Ka) values were obtained from ligand titration experiments at 22 degrees C, pH 7.2. Neither L-arginine nor N alpha-acetyl-L-arginine and N alpha-acetyl-L-arginine methyl ester bind measurably to kringle 5 (Ka approximately less than 0.05 mM-1). In contrast, binding of hexylamine or epsilon-aminocaproic acid (epsilon ACA) is favored (Ka approximately 2.9 and 10.5 mM-1, respectively). Benzamidine and p-benzylaminesulfonic acid associate with kringle 5 with similar affinities (Ka approximately 3.4 and 2.2 mM-1, respectively) while benzylamine binds about twice as tightly (Ka approximately 6.3 mM-1). The higher affinities toward both benzylamine and epsilon ACA indicate that a free carboxylate group is not, by itself, a main determinant of ligand-binding to kringle 5. The experiments also reveal a definite affinity for L-arginine methyl ester, L-lysine, and N alpha-acetyl-L-lysine methyl ester. It is suggested that, although weak (0.1 approximately less than Ka approximately less than 0.6 mM-1), these interactions could be of physiological relevance in the context of plasminogen binding to the fibrin clot. Ligand-induced shifts of kringle 5 proton resonances indicate that the Trp25, His33, Tyr50, Trp62, and Tyr72 (kringle numbering convention) side chains form or neighbor the kringle 5-binding site. Benzamidine-kringle 5 magnetization transfer (Overhauser) experiments verify a close proximity of the bound ligand to these aromatic groups. A model of the binding site is proposed in which the above residues interact closely with each other and define a lipophilic surface which is accessible to the free ligand.     

Water/polymer interactions in a poly(amidoamine) hydrogel studied by NMR spectroscopy

Samples of PAAH1, a cross-linked polymer belonging to the family of poly(amidoamine)s, were investigated at different hydration levels by means of 13C and 1H NMR techniques in order to obtain information on water/polymer interactions. Carbonyl oxygens and amine nitrogens were identified as the main sites of interaction giving hydrogen bonding with water molecules. The polymer turned out to be uniformly plasticized already at moderate degrees of swelling. The hydration process was found to occur in a stepwise manner, with the first batch of water saturating a hydration layer and additional water filling the polymer meshes. The proportion of water in the different states was quantitatively determined.     

Intramolecular interactions, mesomerism and dynamics in actinomycin D studied by 15N NMR spectroscopy

We present a detailed conformational study of 15N-labelled actinomycin D in different organic solvents using 1H, 15N and two-dimensional (2D) NMR techniques at 30.4 MHz and 50.6 MHz. The assignment of the threonine and valine 15N resonances to the individual residues on the alpha- or beta-lactone rings was achieved via heteronuclear shift-correlated 2D NMR experiments. The solvent perturbation studies allow an estimation of the solvent accessibility of the nitrogens and carbonyl groups. Evidence is presented that the pentapeptide rings of actinomycin D have different conformations in polar and in apolar solvents. The chromophoric N10 is efficiently solvent-protected, the solvent-dependence of its 15N resonance resulting from solvent interactions at other positions of the molecule and from solvent-dependent changes in the twisting of the chromophoric system. The chromophoric 2-amino nitrogen is shown to exhibit a strong sp2 character due to the formation of a conjugated system with the carbonyl group at C1. Such a conjugation requires a non-planar chromophoric ring system. Additionally, a hydrogen bond connecting the 2-amino and the 1-carbonyl group was detected. In some solvents, two resonances appear for the 2-amino nitrogen implying the presence of the 2-amino group in two different conformations. The possible implications of the non-planarity of the chromophore for the intercalation process and for the biological activity of the drug are discussed.     

Plastocyanin-cytochrome f interactions: the influence of hydrophobic patch mutations studied by NMR spectroscopy

Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.     

Metabolic characterization of neurological diseases by proton localized NMR spectroscopy of the human brain.

Proton localized Magnetic Resonance Spectroscopy (MRS) of the brain allows the non invasive detection of intracellular cerebral metabolites. Localized MRS has been performed using short stimulated-echo times in various neurological diseases including stroke, multiple sclerosis, and AIDS-related leukoencephalopathies. Principal component analysis (PCA) was used to determine the critical parameters defining the metabolic profile of normal and diseased brain. PCA clearly differentiates the demyelinating processes from ischaemic lesions and leukoencephalopathies. Localized MRS of the brain appears growingly as a tool of choice to discriminate, quantitate and assess cerebral metabolic damage in patients with neurological disorders.     

Structures of lantibiotics studied by NMR

During the last decade, Nuclear Magnetic Resonance has played an important role in the unravelling of the primary and tertiary structures of lantibiotics. A short overview of these studies, together with typical spatial structures obtained, is presented.Abbreviations Ala_S D-alanyl moiety of meso-lanthionine - _SAla L-alanyl moiety of meso-lanthionine - Abu 3-methylalanyl moiety of (2S3S,6R)-3-methyllanthionine - Dha dehydroalanine - Dhb dehydrobutyrine - DPC docecylphosphocholine - NMR Nuclear Magnetic Resonance - NOE Nuclear Overhauser Enhancement - NOESY NOE spectroscopy - TOCSY Total Correlation Spectroscopy     

Mapping protein-protein interactions in solution by NMR spectroscopy.

NMR is very well suited to the study of especially weak protein-protein interactions, as no crystallization is required. The available NMR methods to this end are reviewed and illustrated with applications from the recent biochemical literature: intermolecular NOEs, cross-saturation, chemical shift perturbation, dynamics and exchange perturbation, paramagnetic methods, and dipolar orientation. Most of these methods are now routinely applied for complexes with total molecular mass of 60 kDa and can likely be applied to systems up to 1000 kDa. A substantial fraction of complexes studied show distinct effects of induced fit affecting structural and dynamical properties beyond the contact interface.     

Temperature-reversible eruptions of vesicles in model membranes studied by NMR.

Deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) and freeze-fracture electron microscopy were used to study spontaneous vesiculation in model membranes composed of POPC:POPS with or without cholesterol. The NMR spectra indicated the presence of a central isotropic line, the intensity of which is reversibly and linearly dependent upon temperature in the L alpha phase, with no hysteresis when cycling between higher and lower temperatures. Freeze-fracture microscopy showed small, apparently connected vesicles that were only present when the samples were frozen (for freeze-fracture) from an initial temperature of 40-60 degrees C, and absent when the samples are frozen from an initial temperature of 20 degrees C. Analysis of motional narrowing was consistent with the isotropic lines being due to lateral diffusion in (and tumbling of) small vesicles (diameters approximately 50 nm). These results were interpreted in terms of current theories of shape fluctuations in large unilamellar vesicles which predict that small daughter vesicles may spontaneously "erupt" from larger parent vesicles in order to expel the excess area created by thermal expansion of the bilayer surface at constant volume. Assuming that all the increased area due to increasing temperature is associated with the isotropic lines, the NMR results allowed a novel estimate of the coefficient of area expansion alpha A in multilamellar vesicles (MLVs) which is in good agreement with micromechanical measurements upon giant unilamellar vesicles of similar composition. Experiments performed on unilamellar vesicles, which had been placed upon glass beads, confirmed that alpha A determined in this way is unchanged compared with the MLV case. Addition of the highly positively charged (extrinsic) myelin basic protein (MBP) to a POPC:POPS system showed that membrane eruptions of the type described here occur in response to the presence of this protein.     

Transient protein interactions studied by NMR spectroscopy: the case of cytochrome C and adrenodoxin

The interaction between yeast iso-1-cytochrome c (C102T) and two forms of bovine adrenodoxin, the wild type and a truncated form comprising residues 4-108, has been investigated using a combination of one- and two-dimensional heteronuclear NMR spectroscopy. Chemical shift perturbations and line broadening of amide resonances in the [(15)N,(1)H]HSQC spectrum for both (15)N-labeled cytochrome c and adrenodoxin in the presence of the unlabeled partner protein indicate the formation of a transient complex, with a K(a) of (4 +/- 1) x 10(4) M(-)(1) and a lifetime of <3 ms. The perturbed residues map over a large surface area for both proteins. For cytochrome c, the dominating effects are located around the exposed heme edge but with other areas also affected upon formation of the complex. In the case of adrenodoxin, effects are seen in both the recognition and core domains, with the largest perturbations in the recognition domain. These results indicate that the complex has a dynamic nature, with delocalized binding of cytochrome c on adrenodoxin. A comparison with other transient complexes of redox proteins places this complex between well-defined complexes such as the cytochrome c-cytochrome c peroxidase complex and entirely dynamic complexes such as the cytochrome b(5)-myoglobin complex.