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Affinity chromatography is one way to measure the binding constants of a protein-ligand interaction. Here, we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes.

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses, the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example, the Bmr6 gene in sorghum (Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase (CAD), which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin, enhance digestibility, and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further, many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality., (© 2015 Institute of Botany, Chinese Academy of Sciences.)

Affinity chromatography is one way to measure the binding constants of a protein-ligand interaction. Here we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes.

Switchgrass (Panicum virgatum L.) is being developed as a bioenergy species. Recently an early version of its genome has been released permitting a route to the cloning and analysis of key proteins. Ascorbate peroxidases (APx) are an important part of the antioxidant defense system of plant cells and present a well studied model to understand structure-function relationships. Analysis of the genome indicates that switchgrass encodes several cytosolic ascorbate peroxidases with apparent varying levels of tissue expression. A major cytosolic ascorbate peroxidase was thus selected for further studies. This gene was cloned and expressed in Escherichia coli cells to obtain purified active protein. Full heme incorporation of the enzyme was achieved utilizing slow growth and supplementing the media with 5-aminolevulinic acid. The enzyme was observed to be monomeric in solution via size exclusion chromatography. Activity toward ascorbate was observed that was non-Michaelis-Menten in nature. A site-directed mutant, R172S, was made in an attempt to differentiate activity against ascorbate versus other substrates. The R172S protein exhibited negligible ascorbate peroxidase activity, but showed near wild type activity toward other aromatic substrates., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The challenging nature of studies of membrane proteins has made it difficult to determine the molecular mechanism of transmembrane signaling. For the bacterial chemoreceptor family, there are crystal structures of the internal and external domains, structural models of the transmembrane domain, and evidence for subtle ligand-induced conformational changes, but the signaling mechanism remains controversial. We have used a novel site-directed solid-state NMR distance measurement approach, using (13)C(19)F REDOR, to measure a ligand-induced change of 1.0 +/- 0.3 A in the distance between helices alpha 1 and alpha 4 of the ligand-binding domain in the intact, membrane-bound serine receptor. This distance change is shown not to be due to motion of the side chain and thus is due to motion of either the alpha 1 or the alpha 4 helix. Additional distance measurements can be used to determine the type of backbone motion and to follow it to the cytoplasm, to test and refine current proposals for the mechanism of transmembrane signaling. This is a promising general method for high-resolution measurements of local structure in intact, membrane-bound proteins.

The molecular orientation in a lipid membrane of the peptide fragment VEYAGIALFFVAAVLTLWSMLQYLSAAR (phosphatidylglycerophosphate synthase (Pgs) peptide E) of an integral membrane protein, Pgs, in Escherichia coli has been investigated by solid-state 15N nuclear magnetic resonance (NMR) on macroscopically aligned lipid bilayers. The secondary structure of the peptide in lipid vesicles was determined by circular dichroism spectroscopy. Furthermore, the phase behaviour of the Pgs peptide E/dierucoylphosphatidylcholine (DEruPC)/water system was determined by (2)H, (31)P and 15N solid-state NMR spectroscopy. The phase behaviour obtained was then compared to that of the Pgs peptide E solubilised in dioleoylphosphatidylcholine and water that was previously studied by Morein et al. [Biophys. J. 73 (1997) 3078-3088]. This was aimed to answer the question whether a difference in the length of the hydrophobic part of this peptide and the hydrophobic thickness of the lipid bilayer (hydrophobic mismatch) will affect the phase behaviour. The peptide mostly has a transmembrane orientation and is in an alpha-helical conformation. An isotropic phase is formed in DEruPC with high peptide content (peptide/lipid molar ratio (p/l) > or =1:15) and high water content (> or =50%, w/w) at 35 degrees C. At 55 and 65 degrees C an isotropic phase is induced at high water content (> or =50%, w/w) at all peptide contents studied (no isotropic phase forms in the lipid/water system under the conditions in this study). At high peptide contents (p/l> or =1:15) an isotropic phase forms at 20 and 40% (w/w) of water at 55 and 65 degrees C. A comparison of the phase behaviour of the two homologous lipid systems reveals striking similarities, although the thicknesses of the two lipid bilayers differ by 7 A. This suggests that the rationalisation of the phase behaviour in terms of the hydrophobic mismatch is not applicable to these systems. The C-terminus of Pgs peptide E is amphiphilic and a considerable part of the peptide is situated outside the hydrophobic part of the bilayer, a property of the peptide that to a large extent will affect the lipid/peptide phase behaviour.

The M2 protein from the influenza A virus forms a proton channel in the virion that is essential for infection. This tetrameric protein appears to form a four-helix bundle spanning the viral membrane. Here the solid-state NMR method, 2D polarization inversion spin exchange at magic angle (PISEMA), has been used to obtain multiple constraints from specifically amino acid-labeled samples. The improvement of spectral resolution from 2D PISEMA over 1D methods and 2D separated local field methods is substantial. The reliability of the method is validated by comparison of anisotropic chemical shift and heteronuclear dipolar interactions from single site labeled samples. The quantitative interpretation of the high-resolution constraints confirms the helix tilt to be within the range of previous experimental determinations (32 degrees -38 degrees ). The binding of the channel inhibitor, amantadine, results in no change in the backbone structure at position Val(27,28), which is thought to be a potential binding site for the inhibitor.

Solid-state NMR has been used to study the influence of lipid bilayer hydrophobic thickness on the tilt of a peptide (M2-TMP) representing the transmembrane portion of the M2 protein from influenza A. Using anisotropic (15)N chemical shifts as orientational constraints, single-site isotopically labeled M2-TMPs were studied in hydrated dioleoylphosphatidylcholine (DOPC) and dimyristoylphosphatidylcholine (DMPC) lipid bilayers oriented between thin glass plates. These chemical shifts provide orientational information for the molecular frame with respect to the magnetic field in the laboratory frame. When modeled as a uniform ideal alpha-helix, M2-TMP has a tilt of 37(+/-3) degrees in DMPC and 33(+/-3) degrees in DOPC with respect to the bilayer normal in these lipid environments. The difference in helix tilt between the two environments appears to be small. This lack of a substantial change in tilt further suggests that significant interactions occur between the helices, as in an oligomeric state, to prevent a change in tilt in thicker lipid bilayers., (Copyright 2000 Academic Press.)

The transmembrane portion of the M2 protein from the Influenza A virus has been studied in hydrated dimyristroylphosphotidylcholine lipid bilayers with solid-state NMR. Orientational constraints were obtained from isotopically labeled peptide samples mechanically aligned between thin glass plates. 15N chemical shifts from single site labeled samples constrain the molecular frame with respect to the magnetic field. When these constraints are applied to the peptide, modeled as a uniform alpha-helix, the tilt of the helix with respect to the bilayer normal was determined to be 33 degrees +/- 3 degrees. Furthermore, the orientation about the helix axis was also determined within an error of +/- 30 degrees. These results imply that the packing of this tetrameric protein is in a left-handed four-helix bundle. Only with such a large tilt angle are the hydrophilic residues aligned to the channel axis.