The solution structures of two prophage homologues of the bacteriophage λ Ea8.5 protein reveal a newly discovered hybrid homeodomain/zinc-finger fold.

A cluster of genes in the exoxis region of bacteriophage λ are capable of inhibiting the initiation of DNA synthesis in Escherichia coli. The most indispensible gene in this region is ea8.5.

Lysine methylation strategies for characterizing protein conformations by NMR.

In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino (13)C-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR.

Dioxygen transmembrane distributions and partitioning thermodynamics in lipid bilayers and micelles.

Cellular respiration, mediated by the passive diffusion of oxygen across lipid membranes, is key to many basic cellular processes. In this work, we report the detailed distribution of oxygen across lipid bilayers and examine the thermodynamics of oxygen partitioning via NMR studies of lipids in a small unilamellar vesicle (SUV) morphology. Dissolved oxygen gives rise to paramagnetic chemical shift perturbations and relaxation rate enhancements, both of which report on local oxygen concentration.

Optimizing ¹⁹F NMR protein spectroscopy by fractional biosynthetic labeling.

In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, ¹⁹F and ¹H-¹⁵N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling.

Approaches to the assignment of (19)F resonances from 3-fluorophenylalanine labeled calmodulin using solution state NMR.

Traditional single site replacement mutations (in this case, phenylalanine to tyrosine) were compared with methods which exclusively employ (15)N and (19)F-edited two- and three-dimensional NMR experiments for purposes of assigning (19)F NMR resonances from calmodulin (CaM), biosynthetically labeled with 3-fluorophenylalanine (3-FPhe). The global substitution of 3-FPhe for native phenylalanine was tolerated in CaM as evidenced by a comparison of (1)H-(15)N HSQC spectra and calcium binding assays in the presence and absence of 3-FPhe. The (19)F NMR spectrum reveals six resolved resonances, one of which integrates to three 3-FPhe species, making for a total of eight fluorophenylalanines.

Approaches for the measurement of solvent exposure in proteins by 19F NMR.

Fluorine NMR is a useful tool to probe protein folding, conformation and local topology owing to the sensitivity of the chemical shift to the local electrostatic environment. As an example we make use of (19)F NMR and 3-fluorotyrosine to evaluate the conformation and topology of the tyrosine residues (Tyr-99 and Tyr-138) within the EF-hand motif of the C-terminal domain of calmodulin (CaM) in both the calcium-loaded and calcium-free states. We critically compare approaches to assess topology and solvent exposure via solvent isotope shifts, (19)F spin-lattice relaxation rates, (1)H-(19)F nuclear Overhauser effects, and paramagnetic shifts and relaxation rates from dissolved oxygen.

The measurement of immersion depth and topology of membrane proteins by solution state NMR.

An important component of the study of membrane proteins involves the determination of details associated with protein topology - for example, the location of transmembrane residues, specifics of immersion depth, orientation of the protein in the membrane, and extent of solvent exposure for each residue. Solution state NMR is well suited to the determination of immersion depth with the use of paramagnetic additives designed to give rise to depth-specific relaxation effects or chemical shift perturbations. Such additives include spin labels designed to be "anchored" within a given region of the membrane or small freely diffusing paramagnetic species, whose partitioning properties across the water membrane interface create a gradient of paramagnetic effects which correlate with depth.

Oxygen as a paramagnetic probe of clustering and solvent exposure in folded and unfolded states of an SH3 domain.

The N-terminal SH3 domain of the Drosophila modular protein Drk undergoes slow exchange between a folded (Fexch) and highly populated unfolded (Uexch) state under nondenaturing buffer conditions, enabling both Fexch and Uexch states to be simultaneously monitored. The addition of dissolved oxygen, equilibrated to a partial pressure of either 30 atm or 60 atm, provides the means to study solvent exposure with atomic resolution via 13C NMR paramagnetic shifts in 1H,13C HSQC (heteronuclear single quantum coherence) spectra. Absolute differences in these paramagnetic shifts between the Fexch and Uexch states allow the discrimination of regions of the protein which undergo change in solvent exposure upon unfolding.

19F NMR studies of solvent exposure and peptide binding to an SH3 domain.

(19)F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e.

Tryptophan solvent exposure in folded and unfolded states of an SH3 domain by 19F and 1H NMR.

The isolated N-terminal SH3 domain of the Drosophila signal transduction protein Drk (drkN SH3) is a useful model for the study of residual structure and fluctuating structure in disordered proteins since it exists in slow exchange between a folded (Fexch) and compact unfolded (Uexch) state in roughly equal proportions under nondenaturing conditions. The single tryptophan residue, Trp36, is believed to play a key role in forming a non-native hydrophobic cluster in the Uexch state, with a number of long-range nuclear Overhauser contacts (NOEs) observed primarily to the indole proton. Substitution of Trp36 for 5-fluoro-Trp36 resulted in a substantial shift in the equilibrium to favor the Fexch state.

A combined NMR and molecular dynamics study of the transmembrane solubility and diffusion rate profile of dioxygen in lipid bilayers.

The transmembrane profile of oxygen solubility and diffusivity in a lipid bilayer was assessed by (13)C NMR of the resident lipids (sn-2-perdeuterio-1-myristelaidoyl-2-myristoyl-sn-glycero-3-phosphocholine) in combination with molecular dynamics (MD) simulations. At an oxygen partial pressure of 50 atm, distinct chemical shift perturbations of a paramagnetic origin were observed, spanning a factor of 3.2 within the sn-1 chain and an overall factor of 10 from the headgroup to the hydrophobic interior.

Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins.

This review covers current trends in studies of membrane amphiphiles and membrane proteins using both fast tumbling bicelles and magnetically aligned bicelle media for both solution state and solid state NMR. The fast tumbling bicelles provide a versatile biologically mimetic membrane model, which in many cases is preferable to micelles, both because of the range of lipids and amphiphiles that may be combined and because radius of curvature effects and strain effects common with micelles may be avoided. Drug and small molecule binding and partitioning studies should benefit from their application in fast tumbling bicelles, tailored to mimic specific membranes.

Topology of an outer-membrane enzyme: Measuring oxygen and water contacts in solution NMR studies of PagP.

The topology of the bacterial outer-membrane enzyme, PagP, in dodecylphosphocholine micelles was studied by solution NMR using oxygen and water contacts as probes of hydrophobicity and topology. The effects of oxygen on amide protons were measured at an oxygen partial pressure of 20 atm through the paramagnetic contribution to the relaxation rates associated with the decay of two-spin order. A significant gradation of paramagnetic rates was observed for backbone amides belonging to the transmembrane residues.

Probing the transition state ensemble of a protein folding reaction by pressure-dependent NMR relaxation dispersion.

The F61A/A90G mutant of a redesigned form of apocytochrome b562 folds by an apparent two-state mechanism. We have used the pressure dependence of 15N NMR relaxation dispersion rate profiles to study the changes in volumetric parameters that accompany the folding reaction of this protein at 45 degrees C. The experiments were performed under conditions where the folding/unfolding equilibrium could be studied at each pressure without addition of denaturants.

Nuclear magnetic resonance structures of the zinc finger domain of human DNA polymerase-alpha.

The carboxy terminus of the human DNA polymerase-alpha contains a zinc finger motif. Three-dimensional structures of this motif containing 38 amino acid residues, W L I C E E P T C R N R T R H L P L Q F S R T G P L C P A C M K A T L Q P E, were determined by nuclear magnetic resonance (NMR) spectroscopy. The structures reveal an alpha-helix-like domain at the amino terminus, extending 13 residues from L2 through H15 with an interruption at the sixth residue.

Unwinding of DNA polymerases by the antitumor drug, cis-diamminedichloroplatinum(II).

The helix-turn-helix motifs of the DNA binding domains of human polymerase-alpha and polymerase-kappa are dramatically perturbed upon binding to cisplatin with concomitant release of zinc.

Discovery and biological evaluation of a new family of potent modulators of multidrug resistance: reversal of multidrug resistance of mouse lymphoma cells by new natural jatrophane diterpenoids isolated from Euphorbia species.

The effects of 15 jatrophane diterpene polyesters (1-3 and 5-16) isolated from lipophilic extracts of Euphorbia serrulata, E. esula, E. salicifolia, and E.

[New diterpene polyesters isolated from Hungarian Euphorbia species].

Sixteen new diterpene polyesters were isolated and identified from Hungarian Euphorbiaceae species. Two of them (21, 23) are based on formerly unknown diterpene core. The structures of three jatrophane type diterpene heptaester were elucidated (1, 3, 6), which are diterpenoids with the highest degree of esterification identified from this plant family.

Diterpenoids from Euphorbia peplus.

From a pro-inflammatory active extract of Euphorbia peplus, two new diterpene polyesters based on the pepluane and jatrophane skeletons were isolated, together with four known ingenane and jatrophane diterpenes. The structures were determined on the basis of extensive NMR studies. Ingenol 3-angelate, which was obtained for the first time from this plant, is an irritant toxin with high activity.

A novel lathyrane diterpenoid from the roots of euphorbia lathyris.

A new lathyrane diterpene (1) has been isolated and characterized from a CH2Cl2 extract of the roots of Euphorbia lathyris. Detailed spectral analysis revealed that the structure of 1, including relative stereochemistry, is that of a diester of a hitherto unknown, polyfunctional diterpene parent alcohol.

[Macrocyclic diterpene polyesters of the jatrophane type from Euphorbia esula].

Three new jatrophane diterpenes, esulatin A, B and C (1-3) were isolated and characterized from the whole, undried plant of Euphorbia esula. By means of spectral analysis, the structures were established as penta- and heptaesters of hitherto unknown, polyfunctional diterpene parent alcohols. Esulatin A (1) and C (3) are the diterpenoids with the highest degree of esterification identified to date from the family Euphorbiaceae.