Diacyl phosphatidylglycerols (PG) are the major anionic lipids in the Escherichia coli membrane. Short‒chain dihexanoyl phosphatidylglycerol (DHPG) was previously utilized for the structural determination, by NMR spectroscopy, of the peptide corresponding to the N-terminal membrane anchor of the glucose‒specific enzyme IIA (IIAGlc) from E. coli. This study explores the possible use of lipid micelles of dioctanoyl phosphatidylglycerol (DOPG) and didecanoyl phosphatidylglycerol (DDPG) as alternatives to DHPG. At a peptide concentration of 1 mM, the minimum peptide/lipid molar ratios required for the formation of the lipid‒binding amphipathic helix are approximately 1 :40, 1 :5, and 1 :5 for DHPG, DOPG, and DDPG, respectively. Based on the lipid titration, the critical micelle concentration (CMC) of DHPG was estimated to be ~50 mM. The 1H spectral linewidths of the peptide bound to a variety of lipid micelles decrease in the following order: DDPG &gt; DOPG &gt; DHPG. The helical regions of the peptide in different anionic lipids were elucidated based on chemical shift indexes (CSI). Residues Leu2‒Leu9, Leu2‒Val10, and Leu2‒Val10 were found to be helical in DHPG, DOPG, and DDPG, respectively, indicating that the lipid chain length had only a subtle effect on the amphipathic helix of the peptide. In light of the minimum peptide/lipid ratio and the spectral linewidth, and the CSI‒derived peptide structure, DOPG is proposed as a good compromise for structural studies of this membrane‒associated peptide by solution NMR spectroscopy.

Guangshun Wang

Paul A. Keifer

Alan Peterkofsky

Short‒chain diacyl phosphatidylglycerols: which one to choose for the NMR structural determination of a membrane‒associated peptide from Escherichia coli?

The recombination activating gene products (RAG1 and 2) catalyze the initial DNA cleavage steps during V(D)J recombination for the diversification of the immune repertoire. As the fundamental properties of the RAG proteins remain largely unknown, our objective is to investigate the self&#8211;association and conformational properties of RAG1. To analyze RAG1 association and dissociation, a time course of multi&#8211;angle laser light scattering measurements (MALL SEC) was performed on samples at different oligomerization states over a wide range of ionic strengths. The molecular masses of the predominant RAG1 species corresponded to dimer, tetramer, and a previously uncharacterized octamer state. Furthermore, the fraction of RAG1 in the tetrameric and octameric states increased significantly over time at lower ionic strengths, indicating that these oligomeric forms may factor into the physiological function of RAG1. Circular dichroism (CD) analysis of RAG1 showed a significant dependence in secondary structure on ionic strength with changes in &#945;-helical content over time that may correspond to the changes in oligomerization states shown by MALLS SEC. Together, the MALLS SEC and CD analyses of RAG1 self-association properties and secondary structure give further insight into formation of the protein complex responsible for catalyzing V(D)J recombination.

LeAnn J. Godderz

Karla K. Rodgers

RAG1 oligomerization states and secondary structural properties: an initial characterization of V(D)J recombinase complex formation

Metal polypyridines are excellent candidates for gas-phase optical experiments where their intrinsic properties can be studied without complications due to the presence of solvent. The fluorescence lifetimes of [Ru(bpy)3]1&#x2b; trapped in an optical detection cell within a Fourier transform mass spectrometer were obtained using matrix-assisted laser desorption/ionization to generate the ions with either 2,5-dihydroxybenzoic acid (DHB) or sinapinic acid (SA) as matrix. All transients acquired, whether using DHB or SA for ion generation, were best described as approximately exponential decays. The rate constant for transients derived using DHB as matrix was 4&#215;107 s&#8722;1, while the rate constant using SA was 1&#215;107 s&#8722;1. Some suggestions of multiple exponential decay were evident although limited by the quality of the signals. Photodissociation experiments revealed that [Ru(bpy)3]1&#x2b; generated using DHB can decompose to [Ru(bpy)2]1&#x2b;, whereas ions generated using SA showed no decomposition. Comparison of the mass spectra with the fluorescence lifetimes illustrates the promise of incorporating optical detection with trapped ion mass spectrometry techniques.

Jill R. Scott

Jason E. Ham

Bill Durham

Paul L. Tremblay

Ruthenium trisbipyridine as a candidate for gas-phase spectroscopic studies in a Fourier transform mass spectrometer

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyses the formation of pyroglutamyl residues from glutamine at the N-terminus of peptides and proteins. In previously applied assays, QC activity was determined by either analysing the products formed using HPLC coupled with photometric or fluorometric detection, radioimmunoassay, or by detecting the release of ammonia spectrophotometrically. Although these methods are sensitive, they are all discontinuous and therefore time-consuming and laborious. To conduct a detailed kinetic investigation of QC catalysis, we developed coupled continuous assays suitable for microplates which allow now convenient determination of QC activity. The methods either use pyroglutamyl aminopeptidase or glutamate dehydrogenase as auxiliary enzymes, which results in the liberation of chromophores or fluorophores such as pNA, AMC, &#946;NA or in the conversion of the chromophore NADH/H&#x2b; into NAD&#x2b;, respectively.The assays were applied in various enzyme isolation and characterisation studies, using crude protein solutions as well as purified enzyme in pH-dependence, substrate and inhibitor specificity investigations. Depending on the respective analytical task, both assays complement each other. Therefore, different enzymatic properties could be explored in more detail. Since the employed strategy of assay development could be of interest also for the analysis of other enzymes, the methods are described here in a comprehensive manner.

Stephan Schilling

Hans-Ulrich Demuth

Continuous assays of glutaminyl cyclase: from development to application

Along with significant progress in low-frequency electron spin resonance (ESR, also called electron paramagnetic resonance, EPR), other techniques such as Longitudinally-Detected ESR (LODESR), proton electron double-resonance imaging (PEDRI) and field-cycled dynamic nuclear polarization (FC-DNP) have been developed for in vivo applications. However their potential is still far from maximally defined, in part, because of the need for new specific function-directed spin probes. An application of stable nitroxides of imidazoline and imidazolidine types provides unique possibility to measure local values of pH and thiol content in various biological systems, including in vivo studies. These applications are based on the observation of specific chemical reactions of these nitroxides with protons or thiols, followed by significant changes in their EPR spectra. To increase sensitivity of pH probes for low-frequency EPR spectroscopy we evaluated two alternative approaches: (i) application of isotopically substituted labels, and (ii) acquisition of EPR spectra at high modulation amplitude. Spatial and spectral-spatial imaging (pH-mapping) using PEDRI and L-band EPR imagers were performed both on phantom samples and in vivo. The applications of the pH and SH probes in model systems, biological fluids, and in vivo in living animals are discussed.

Valery V. Khramtsov

Igor A. Grigor&#x2019;ev

David J. Lurie

Margaret A. Foster

Jay L. Zweier

Periannan Kuppusamy

Spin pH and SH probes: enhancing functionality of EPR-based techniques

High field 1H NMR spectroscopy has been employed to obtain, in conjunction with chemometric analysis, information regarding fluctuations in endogenous metabolic profiles for Crotalaria cobalticola plant cells following exposure to cobalt chloride.Such ‘metabolomic’ type data analysis is often confounded by experimental, environmental or genetic factors that are not correlated to the classifications of interest and serve only to complicate the extraction of meaningful answers from a dataset. This work demonstrates the application of data filtering to remove extraneous data that result from spectrometer variation rather than being correlated with the classes of interest. Samples were analysed from Crotalaria cobalticola suspension cell culture following exposure to cobalt chloride using 2 spectrometers. Removal of confounding data due to spectrometer variation resulted in clear separation between control and dosed classes. It was then possible to use the model to determine key changes in biochemical status caused as a result of exposure to cobalt. Branched chain amino acids, succinate and secondary metabolite precursors phenylalanine and tyrosine were all higher in the control samples, whilst choline, glutamate, alanine and lactate were higher in the dosed samples.

Nigel J. C. Bailey

Matjaz Oven

Elaine Holmes

Meinhart H. Zenk

Jeremy K. Nicholson

An NMR-based metabolomic approach to the analysis of the effects of xenobiotics on endogenous metabolite levels in plants

Oxidative modification of proteins can interfere with critical cellular functions, and is widely regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to atherosclerosis and cancer. In this line, a new GC‒MS methodology using N(O)‒ethoxycarbonyl trifluoroethyl amino acid esters (ECEE‒F3) for rapid and sensitive determination of 3‒chlorotyrosine, 5‒hydroxy‒2‒aminovaleric acid (HAVA), and 6‒hydroxy‒2‒aminocaproic acid (HACA) in proteins has been developed. 3‒Chlorotyrosine is a highly specific marker of myeloperoxidase catalyzed protein oxidation, whereas &#x3B3;‒glutamyl semialdehyde (&#x3B3;GSA) and &#x03B1;‒aminoadipyl semialdehyde (&#x03B1;ASA), which by reduction form HAVA and HACA, respectively, are specifically formed by metal catalyzed oxidation processes. ECEE‒F3 derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. The key steps of the methodology employed are (i) enzymatic hydrolysis of target proteins to prevent decomposition of oxidation products during hydrolysis and (ii) an uniquely rapid derivatization of amino acids completing sample preparation for GC within a few minutes in aqueous solution at room temperature. The use of these stable products of protein amino acid side chain oxidation as potential markers for assessing oxidative damage in LDL apoB‒100 recovered from human aortic vascular lesions is demonstrated. These observations provide quantitative chemical evidence for metal catalyzed oxidative processes in the human artery wall.

Jens Pietzsch

Ralf Bergmann

Steffi Kopprasch

Analysis of non-protein amino acids as specific markers of low density lipoprotein apolipoprotein B-100 oxidation in human atherosclerotic lesions: the use of N(O)-ethoxycarbonyl trifluoroethyl ester derivatives and GC-MS 1

This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations of metal effects upon peptide–receptor interactions with the use of NMR methods. Cerium(III) is herein used for assessing metal effects upon the interaction between angiotensin II and a fragment from the AT1A receptor. Angiotensin II forms a complex with cerium(III) in water while the fCT300–320 receptor fragment is poorly affected by cerium(III). However, the addition of the fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for the receptor and probing the peptide residues involved in receptor binding.

Nicola D'Amelio

Elena Gaggelli

Nicola Gaggelli

Francesca Mancini

Elena Molteni

Daniela Valensin

Gianni Valensin

Probing the role of metal ions on reversible peptide–protein interactions by NMR

Calpains are intracellular cysteine endopeptidases that combine protease activity with a dependence on Ca2&#x2b; binding. Here we describe the conformational changes leading to the calpain activation, occurring in the enzyme purified from human erythrocytes, studied in solution using small angle X-ray scattering (SAXS). Addition of Ca2&#x2b; determines the formation of large soluble aggregates that can be dissociated either chelating these ions or adding cahotropic salts in solution. On the other side, our SAXS studies revealed that the addition of Ca2&#x2b; in the presence of inhibitors as E64 or leupeptin triggers a reversible conformational transition leading to a proper assembly of the active site without any aggregation or dissociation process. It is suggested that the observed conformational changes can be considered as the early step in the sequence of molecular events leading to calpain activation.

Enrico Dainese

Annalaura Sabatucci

Roberto Minafra

Patrice Vachette

Edon Melloni

Ivo Cozzani

Molecular events involved in the activation of calpain from human erythrocytes

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Journal of Spectroscopy publishes research into the theory and application of spectroscopy across all disciplines, including biology, chemistry, engineering, earth sciences, medicine, materials science, physics, and space science.

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Chief Editor&nbsp;Dr Daniel Cozzolino&nbsp;is based at the University of Queensland, Australia. His research focuses on the developments of chemometric and spectroscopic methods for use in agriculture and food applications.

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