Mapping protein–protein interaction by 13C′-detected heteronuclear NMR spectroscopy

The copper-mediated protein–protein interaction between yeast Atx1 and Ccc2 has been examined by protonless heteronuclear NMR and compared with the already available ¹H–¹⁵N HSQC information. The observed chemical shift variations are analyzed with respect to the actual solution structure, available through intermolecular NOEs. The advantage of using the CON-IPAP spectrum with respect to the ¹H–¹⁵N HSQC resides in the increased number of signals observed, including those of prolines. CBCACO-IPAP experiments allow us to focus on the interaction region and on side-chain carbonyls, while a newly designed CEN-IPAP experiment on side-chains of lysines. An attempt is made to rationalize the chemical shift variations on the basis of the structural data involving the interface between the proteins and the nearby regions. It is here proposed that protonless ¹³C direct-detection NMR is a useful complement to ¹H based NMR spectroscopy for monitoring protein–protein and protein–ligand interactions. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at     

Folding in solution of the C‐catalytic protein fragment of angiotensin‐converting enzyme

Angiotensin‐converting enzyme (ACE) is a key molecule of the renin–angiotensin–aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala₉₅₉ to Ser₁₀₆₆ region (ACE_C) that corresponds to the C‐catalytic domain of human somatic angiotensin‐I‐converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1‐trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE_959–1066 protein fragment in order to study its structure in solution by NMR spectroscopy. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational dynamics of the anthrax lethal factor catalytic center

Anthrax lethal factor (LF) is a zinc-metalloprotease that together with the protective antigen constitutes anthrax lethal toxin, which is the most prominent virulence factor of the anthrax disease. The solution nuclear magnetic resonance and in silico conformational dynamics of the 105 C-terminal residues of the LF catalytic core domain in its apo form are described here. The polypeptide adopts a compact structure even in the absence of the Zn(2+) cofactor, while the 40 N-terminal residues comprising the metal ligands and residues that participate in substrate and inhibitor recognition exhibit more flexibility than the C-terminal region.     

An overview of models of stomatal conductance at the leaf level

Stomata play a key role in plant adaptation to changing environmental conditions as they control both water losses and CO₂ uptake. Particularly, in the context of global change, simulations of the consequences of drought on crop plants are needed to design more efficient and water‐saving cropping systems. However, most of the models of stomatal conductance (g_s) developed at the leaf level link g_s to environmental factors or net photosynthesis (A_net), but do not include satisfactorily the effects of drought, impairing our capacity to simulate plant functioning in conditions of limited water supply. The objective of this review was to draw an up‐to‐date picture of the g_s models, from the empirical to the process‐based ones, along with their mechanistic or deterministic bases. It focuses on models capable to account for multiple environmental influences with emphasis on drought conditions. We examine how models that have been proposed for well‐watered conditions can be combined with those specifically designed to deal with drought conditions. Ideas for future improvements of g_s models are discussed: the issue of co‐regulation of g_s and A_net; the roles of CO₂, absissic acid and H₂O₂; and finally, how to better address the new challenges arising from the issue of global change.     

Evolution of xyloglucan-related genes in green plants

Background  

The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.     

Structure-function discrimination of the N- and C- catalytic domains of human angiotensin-converting enzyme: implications for Cl- activation and peptide hydrolysis mechanisms

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two sequence homologous protein domains (ACE_N and ACE_C) possessing several biochemical features that differentiate the two active sites (i.e. chloride ion activation). Based on the recently solved X-ray structure of testis angiotensin-converting enzyme (tACE), the 3D structure of ACE_N was modeled. Electrostatic potential calculations reveal that the ACE_N binding groove is significantly more positively charged than the ACE_C, which provides a first rationalization for their functional discrimination. The chloride ion pore for Cl2 (one of the two chloride ions revealed in the X-ray structure of tACE) that connects the external solution with the inner part of the protein was identified on the basis of an extended network of water molecules. Comparison of ACE_C with the X-ray structure of the prokaryotic ClC Cl(-) channel from Salmonella enterica serovar typhimurium demonstrates a common molecular basis of anion selectivity. The critical role for Cl2 as an ionic switch is emphasized. Sequence and structural comparison between ACE_N and ACE_C and of other proteins of the gluzincin family highlights key residues that could be responsible for the peptide hydrolysis mechanism. Currently available mutational and substrate hydrolysis data for both domains are evaluated and are consistent with the predicted model.     

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

NMR spectroscopy was used to study the effect of guanidinium chloride on the unfolding of horse heart and yeast iso-1 cytochrome c under mild alkaline conditions. The structural changes on the horse heart protein were detected through NOESY (Nuclear Overhauser Effect SpectroscopY) experiments whereas (15)N-(1)H heteronuclear NMR was used to monitor the behavior of the yeast protein. The latter represents the first characterization through (15)N-(1)H heteronuclear NMR spectroscopy of the guanidinium chloride induced unfolding of mitochondrial cytochrome c. The presence of denaturants decreases the temperature at which the native Met80 axial ligand is displaced from the iron center under the present mild alkaline conditions. The process can be described in terms of protein fragments behaving as unfolding units of different stability. The comparison between the two proteins indicates that the loop+helix connecting the proximal and distal sites, as well as the long Met80-containing loop immediately after a short helix, are structural characteristics of mitochondrial cytochrome c that appear to be responsible for the Met80-iron(III) bond fragility.     

Solution structure of reduced horse heart cytochrome c

 In the frame of a broad study on the structural differences between the two redox forms of cytochromes to be related to the electron transfer process, the NMR solution structure of horse heart cytochrome c in the reduced form has been determined. The structural data obtained in the present work are compared to those already available in the literature on the same protein and the presence of conformational differences is discussed in the light of the experimental method employed for the structure determination. Redox-state dependent changes are analyzed and in particular they are related to the role of propionate-7 of the heme. Also some hydrogen bonds are changed upon reduction of the heme iron. A substantial similarity is observed for the backbone fold, independently of the oxidation state. At variance, some meaningful differences are observed in the orientation of a few side chains. These changes are related to those found in the case of the highly homologous cytochrome c from Saccharomyces cerevisiae. The exchangeability of the NH protons has been investigated and found to be smaller than in the case of the oxidized protein. We think that this is a characteristic of reduced cytochromes and that mobility is a medium for molecular recognition in vivo. Received: 8 June 1998 / Accepted: 13 October 1998     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.