We have analyzed the adsorption of protein to the surfaces of silica nanoparticles with diameters of 6, 9, and 15 nm. The effects upon adsorption on variants of human carbonic anhydrase with differing conformational stabilities have been monitored using methods that give complementary information, i.e., circular dichroism (CD), nuclear magnetic resonance (NMR), analytical ultracentrifugation (AUC), and gel permeation chromatography. Human carbonic anhydrase I (HCAI), which is the most stable of the protein variants, establishes a dynamic equilibrium between bound and unbound protein following mixture with silica particles. Gel permeation and AUC experiments indicate that the residence time of HCAI is on the order of approximately 10 min and slowly increases with time, which allows us to study the effects of the interaction with the solid surface on the protein structure in more detail than would be possible for a process with faster kinetics. The effects on the protein conformation from the interaction have been characterized using CD and NMR measurements. This study shows that differences in particle curvature strongly influence the amount of the protein's secondary structure that is perturbed. Particles with a longer diameter allow formation of larger particle-protein interaction surfaces and cause larger perturbations of the protein's secondary structure upon interaction. In contrast, the effects on the tertiary structure seem to be independent of the particles' curvature.

Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula 3 tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up-and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.

No abstract

Phenol hydroxylase from Pseudomonas sp. CF600 is a member of a family of binuclear iron-center-containing multicomponent oxygenases, which catalyzes the conversion of phenol and some of its methyl-substituted derivatives to catechol. In addition to a reductase component which transfers electrons from NADH, optimal turnover of the hydroxylase requires P2, a protein containing 90 amino acids which is readily resolved from the other components. The three-dimensional solution structure of P2 has been solved by 3D heteronuclear NMR spectroscopy. On the basis of 1206 experimental constraints, including 1060 distance constraints obtained from NOEs, 70 phi dihedral angle constraints, 42 psi dihedral angle constraints, and 34 hydrogen bond constraints, a total of 12 converged structures were obtained. The atomic root mean square deviation for the 12 converged structure with respect to the mean coordinates is 2.48 A for the backbone atoms and 3.85 A for all the heavy atoms. This relatively large uncertainty can be ascribed to conformational flexibility and exchange. The molecular structure of P2 is composed of three helices, six antiparallel beta-strands, one beta-hairpin, and some less ordered regions. This is the first structure among the known multicomponent oxygenases. On the basis of the three-dimensional structure of P2, sequence comparisons with similar proteins from other multicomponent oxygenases suggested that all of these proteins may have a conserved structure in the core regions.

Identification of compounds from chemical libraries that bind to macromolecules by use of NMR spectroscopy has gained increasing importance during recent years. A simple methodology based on (19)F NMR spectroscopy for the screening of ligands that bind to proteins, which also provides qualitative information about relative binding strengths and the presence of multiple binding sites, is presented here. A library of fluorinated compounds was assembled and investigated for binding to the two bacterial chaperones PapD and FimC, and also to human serum albumin (HSA). It was found that library members which are bound to a target protein could be identified directly from line broadening and/or induced chemical shifts in a single, one-dimensional (19)F NMR spectrum. The results obtained for binding to PapD using (19)F NMR spectroscopy agreed well with independent studies based on surface plasmon resonance, providing support for the versatility and accuracy of the technique. When the library was titrated to a solution of PapD chemical shift and linewidth changes were observed with increasing ligand concentration, which indicated the presence of several binding sites on PapD and enabled the assessment of relative binding strengths for the different ligands. Screening by (19)F NMR spectroscopy should thus be a valuable addition to existing NMR techniques for evaluation of chemical libraries in bioorganic and medicinal chemistry.

No abstract

Mercuric ions are toxic to living organisms because of their strong affinity for cysteine residues in proteins. Some bacteria have developed a resistance mechanism whereby Hg2+ is transported into the cytoplasm and reduced to Hg0. One of the proteins involved in the transport of mercuric ion is the periplasmic binding protein MerP, which can exist both as oxidized (disulfide) and as reduced (dithiol) forms. Only the reduced form with Cys-17 and Cys-14 residues as free thiols is a potent receptor for mercuric ion. In this work the solution structure of the oxidized form of MerP has been determined by multidimensional NMR spectroscopy and compared to the NMR structures of the previously published structures of the reduced and mercury-bound forms of MerP. The mercury-bound and oxidized forms have similar tertiary structures, whereas in the reduced form there is a large rearrangement of the mercuric ion binding loop and the nearby loop comprising residues 38-41. The structural arrangement of the latter loop seems to be important for the stabilization of the surface location of the cysteine-containing loop. In the reduced form at low pH the cysteine-containing loop adopts a conformation similar to what is observed in the oxidized and mercury-bound forms. The oxidized form also differs with respect to the other two forms in the relative positions of some of the alpha-helices and beta-strands. Structural differences between the oxidized and reduced forms may help explain why the reduced form is stable in the periplasm, which is considered to be an oxidizing environment.

The binding of protein to solid surfaces often induces changes in the structure, and to investigate these matters we have selected two different protein-nanoparticle systems. The first system concerns the enzyme human carbonic anhydrase II which binds essentially irreversibly to the nanoparticles, and the second system concerns human carbonic anhydrase I which alternate between the adsorbed and free state upon interaction with nanoparticles. Application of the TROSY pulse sequence has allowed high-resolution NMR analysis for both of the protein-nanoparticle systems. For HCAII it was possible to observe spectra of protein when bound to the nanoparticles. The results indicated that HCAII undergoes large rearrangements, forming an ensemble of molten globule-like structures on the surface. The spectra from the HCAI-nanoparticle system are dominated by HCAI molecules in solution. A comparative analysis of variations in intensity from 97 amide resonances in a 1H-15N TROSY spectrum revealed the effects from interaction with nanoparticle on the protein structure at amino acid resolution.