Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-Å resolution. The enzyme folds into a right-handed parallel b-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long a-helices aligned in an antiparallel manner in a classical up-and-down fourhelical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel b-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.

In living organisms, genes encoding proteins that contain flavins as a prosthetic group constitute approximately 2–3% of the total. The fluorescence of flavin cofactors in these proteins is a property that is widely employed for biochemical characterisation. Here, we present a modified Thermofluor® approach called ThermoFAD (Thermofluor®‐adapted flavin ad hoc detection system), which simplifies identification of optimal purification and storage conditions as well as high‐affinity ligands. In this technique, the flavin cofactor is used as an intrinsic probe to monitor protein folding and stability, taking advantage of the different fluorescent properties of flavin‐containing proteins between the folded and denatured state. The main advantage of the method is that it allows a large amount of biochemical data to be obtained using very small amounts of protein sample and standard laboratory equipment. We have explored several cases that demonstrate the reliability and versatility of this technique when applied to globular flavoenzymes, membrane‐anchored flavoproteins, and macromolecular complexes. The information gathered from ThermoFAD analysis can be very valuable for any biochemical and biophysical analysis, including crystallisation. The method is likely to be applicable to other classes of proteins that possess endogenous fluorescent cofactors and prosthetic groups.

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c 552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c 552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c 551 , which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.

(8,9). A number of investigators have found that the level of MAO B in preparations exhibiting high affinity sites is only 5-10% of the total enzyme present (9 -11). It is not known whether this high affinity binding also results in enzyme inhibition. To date, no molecular explanation has been found to resolve these observations except to propose that the nanomolar binding site is separate from the active site and is possessed by a subpopulation of enzyme occurring by an unknown mechanism. No evidence exists for any altered enzyme forms (alternate splicing or posttranslational modification(s)), which might account for the observed substoichiometric levels of high affinity binding sites on MAO B.The Eli Lilly group (11-13) observed that inhibition of human MAO B by tranylcypromine increases the level of high affinity I 2 -binding sites from 5-10% to ϳ90% of the total enzyme. This potentiation is observed with MAO B in human platelets, in membrane preparations from human cortex, and from medulla preparations as well as with membrane particles of human recombinant MAO B (but not with human MAO A) expressed in insect cells (13). These data suggest that inhibition of MAO B by tranylcypromine alters the enzyme to a form that * This work was supported, in whole or in part, by National Institutes of Health Grant GM 29433 (to D. E. E.) and a predoctoral fellowship from the National Institutes of Health through NINDS Award F31NS063648 (to E. M. M.). This work was also supported by grants from the Fondazione Cariplo (to D. E. E. and A. M.), , and Canadian Institutes of Health Research (Grant MOP77529) to A. H. The atomic coordinates and structure factors (codes 2XFU, 2XCG, 2XFN, 2XFO, 2XFP, and 2XFQ) The abbreviations used are: MAO, monoamine oxidase; 2-BFI, 2-(2-benzofuranyl)-2-imidazoline.

No abstract