br Protein molecular evolution analysis The protocol

Protein molecular evolution analysis The protocol included new protein molecular evolution tests integrating patterns of nucleotide sequence similarities with protein tertiary structures. The MEGA program was used in calculations of codon usage statistics. Specifically, the ratios between observed and expected amino erk inhibitors codon counts determined relative synonymous codon usage statistics (R) that indicated amino acid codons with R≤0.7 as not preferable amino acid codons. In reference protein amino acid sequences, there were invariant amino acid sites (invariant alignment positions), forward amino acid sites (variant alignment positions that did not include not preferable amino acid codons) and compensatory amino acid sites (variant alignment positions that included not preferable amino acid codons). The presence of preferable amino acid codons, as well as absence of not preferable amino acid codons indicated that forward amino acid sites could have major influence on protein tertiary structures and functions. The DeepView/Swiss-PdbViever was used in analyses of protein tertiary structures (http://spdbv.vital-it.ch/).
Acknowledgements
Data We have extracted a total of 2089 NOE distance restraints from three-dimensional 15N-edited and 13C-edited NOESY spectra, which were processed using NMRPipe [1,2]. Spectra exhibit substantial chemical shift dispersion – a feature also observed for the one-dimensional 1H NMR spectrum of CupS (Fig. 1). In total, this data set consists of 929 intra-residual, 448 sequential, 281 medium range, and 431 long-range NOE distance restraints, supplemented by 221 NMR-derived dihedral angle restraints from TALOS+ [3]. These experimental restraints are compatible with the software suite ARIA 2.3 [4]/CNS 1.2.1 [5,6]. NOEs were picked manually and obvious intraresidual and sequential NOEs were assigned hand-operated. ARIA2.3 [4]/CNS 1.2.1 [5,6] and UNIO (ATNOS/CANDID) [7]/CYANA 3.0 [8] software packages were used to automatically assign the picked NOE resonances.
Experimental design, materials and methods
Acknowledgements Part of this work was performed under the Cooperative Research Program of the Institute for Protein Research, Osaka University, with a lot of help from members of the Prof. Genji Kurisu׳s group in IPR. This work was supported by the RUB Research School (DFG GSC 98/3) to H.W. and by a grant of the Deutsche Forschungsgemeinschaft DFG to M.M.N. (No. 836/1-1).
Introduction Human immunodeficiency virus type 1 (HIV-1) infects cells through interaction with the CD4 receptor and one of two chemokine coreceptors, either CCR5 or CXCR4 [1]. Approximately 80–90% of recently infected and treatment-naïve HIV-1 patients have a virus that uses the CCR5 coreceptor (R5 virus) [2], while the CXCR4 coreceptor-using virus (X4 virus) and dual-tropic viruses that use both CCR5 and CXCR4 (R5×4) emerge and coexist in nearly half of non-treated subtype B and D infected patients in advanced disease stages but are found less often in subtype A and C infected individuals [3]. The development of the CCR5 coreceptor blocker maraviroc, which has exclusive activity against R5 viruses, has attracted considerable attention in co-receptor affinity or tropism [4]. It is strongly recommended that we determine co-receptor tropism before initiating treatment with entry inhibitors because these drugs have no effect on X4 populations [5]. One of the principal approaches to assess HIV-1 coreceptor use is the genotypic assay, which infers viral tropism from sequence information of the third hypervariable (V3) loop of gp120 in the envelope protein. This approach comprises two steps: the sequencing assay, and the prediction method interpreting the sequence data. Sequencing is usually based on the conventional “bulk” Sanger sequencing method. This approach is neither ideal for sequencing DNA that contains nucleotide mixtures (quasispecies) nor DNA with mutations being present in at least 15–20% of the viral population. Most of these limitations have now been overcome by the introduction of next generation sequencing. This technology allows, for instance, sequencing not only single clones but also viruses prevalent in minor populations [6–8]. Despite the larger number of methods developed for predicting HIV-1 coreceptor use, only a small number of these algorithms are implemented as web-service. Among the available web-tools, geno2-pheno co-receptor (g2p) is the most used algorithm applied to Support Vector Machines (SVMs) to infer coreceptor use. Unlike other methods, gen2pheno, for example, can handle massively parallel sequencing [MPS] data and allows for changing the specificity-level of the prediction method.