NMR solution structure of a cold-adapted thiol-disulfide oxidoreductase

Psychrophilic enzymes produced by cold-adapted micro-organisms have successfully overcome the low temperature challenge and adapted to maintain high catalytic rates in their permanently cold environments. The current consensus is that this high activity at low temperatures is mainly achieved through an increase in the flexibility of the protein structure, thereby allowing for the molecular motions necessary for activity in their low thermal energy environment. The actual molecular basis for the adaptation is still however only poorly understood and direct evidence of the proposed increased flexibility is scant, with previous attempts to demonstrate this leading to conflicting results. In an attempt to better understand strategies of cold adaptation we have determined the NMR solution structure of the reduced form of a cold adapted thiol disulphide oxidoreductase (DsbA) isolated from an Antarctic bacterium. While a number of crystal structures for cold adapted enzymes have been published, this is the first report of an NMR structure of these enzymes and thereby opens up a new dimension in the study of cold adaptation. In particular, the potential power of NMR to monitor both local and global motions over a large range of time scales should allow for a better understanding of the role of dynamics in protein adaptation to temperature. The gene encoding the cold-adapted enzyme has been isolated and the protein overexpressed in E. coli with both unlabelled and labelled (15N13C, 15N) protein being purified from the periplasmic extracts. NMR data were acquired on a Bruker AvanceII+ 800 MHz spectrometer and the solution structure of the reduced form of this cold adapted oxidoreductase determined and compared to that of its mesophilic homolog from Vibrio cholerae. In addition, the temperature dependence of activity and stability of both the psychrophile and mesophile have been ascertained and compared. Here, the results of the NMR structure determination and the comparative structural and physicochemical studies of the cold adapted DsbA with its mesophilic homolog will be presented.European Molecular Biology Organisation (EMBO). Fundação para a Ciência e a Tecnologia (FCT) via Programa Ciência 2008

Backbone and side chain 1H, 15N and 13C assignments for a thiol-disulphide oxidoreductase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Enzymes produced by psychrophilic organisms have successfully overcome the low temperature challenge and evolved to maintain high catalytic rates in their permanently cold environments. As an initial step in our attempt to elucidate the cold-adaptation strategies used by these enzymes we report here the 1H, 15N and 13C assignments for the reduced form of a thiol-disulphide oxidoreductase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.The NMR spectrometers are part of The National NMR Network (REDE/1517/RMN/2005), supported by ‘‘Programa Operacional Ciência e Inovação (POCTI) 2010’’ and Fundação para a Ciência e a Tecnologia (FCT). This work was funded by FCT, POCTI and FEDER; Projects POCI/BIA-PRO/57263/2004 and PTDC/BIO/70806/2006. TC is holder of a long term EMBO fellowship. MM is thankful to the Fundação para a Ciência e Tecnologia for its support through Programa Ciência 2007.info:eu-repo/semantics/publishedVersio

Urinary proteome and metabolome in dogs (Canis lupus familiaris): The effect of chronic kidney disease

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands <65 kDa) proteinuria. Urine spectra acquired by NMR allowed the identification of 86 metabolites in healthy dogs, belonging to 49 different pathways mainly involved in amino acid metabolism, purine and aminoacyl-tRNA biosynthesis or tricarboxylic acid cycle. Seventeen metabolites showed significantly different concentrations when comparing healthy and CKD dogs. In particular, carnosine, trigonelline, and cis-aconitate, might be suggested as putative biomarkers of CKD in dogsinfo:eu-repo/semantics/acceptedVersio

Structural Characterization by NMR of a Double Phosphorylated Chimeric Peptide Vaccine for Treatment of Alzheimer’s Disease

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer’s disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229-237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241-255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible

Hydrolytic Zinc Metallopeptides Using a Computational Multi-State Design Approach

International audienc

The solution structure of the soluble form of the lipid-modified azurin from Neisseria gonorrhoeae, the electron donor of cytochrome c peroxidase

We thank Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided to SRP (PTDC/BIA-PRO/109796/2009), CSN (SFRH/BD/87878/2012) and IHS (SFRH/BPD/84404/2012), and that support the 600 MHz and 800 MHz NMR spectrometers that are part of the National NMR Network (RECI/BBB-BQB/0230/2012).Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277 +/- 5 mV, at pH 7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the beta-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment. (C) 2015 Elsevier B.V. All rights reserved.publishersversionpublishe