br Sub confluent cultures of HCT cells were

Sub-confluent cultures of HCT116 cells were transiently transfected with phCL-EGFP yielding expression of full-length cathepsin L, which was co-localized with endogenous cathepsin L and also reached the nuclei (see Fig. 7D′ in [1]). In addition, MTT assays were performed with non-transfected, pEGFP-N1-, and phCL-EGFP-transfected HCT116 cells after 24h. Data revealed that proliferation rates were not altered upon expression of enhanced GFP alone, while the MTT conversion rates were increased in HCT116 cells expressing hCL-EGFP chimeras (Fig. 5), although this increase did not reach significance over non-transfected controls.
Molecular forms of cathepsin L-EGFP chimeras translated in HCT116 cells To identify the molecular forms of the cathepsin L-EGFP chimeras translated in HCT116 cells, immunoblotting was performed with whole cell Iysates of non-transfected cultures and with those expressing pEGFP-N1 or phCL-EGFP, respectively. The expected molecular forms of cathepsin L were detected in all samples, i.e. the pro-form as well as the single chain (SC) and heavy chain (HC) of the two-chain form of the protease. Lysates of phCL-EGFP-transfected HCT116 cells featured an additional band at 65kDa that was recognized by cathepsin L-specific SCR7 (Fig. 6), and which was absent from non-transfected cells or those transfected with the empty vector pEGFP-N1. The data indicates that the band at 65kDa is representative of the hCL-EGFP chimeric protein which was stable for 24h in HCT116 cells because no degradation bands could be observed in immunoblots with cathepsin L-specific antibodies (Fig. 6).
Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (DFG), Grant BR 1308/6-1, 6-2, and 10-1 to KBr, and by the South-East Norwegian Regional Health Authorities HSØ, Grant #2011142, to MHH and GMM. MHH was also supported by a stipend from the Deutscher Akademischer Austauschdienst DAAD, A/11/95548.
Data
Experimental design, materials and methods
Competing financial interests
Specification Table
Value of the data
Data, experimental design, materials and methods
Acknowledgements This work was supported by research grant from the Natural Sciences and Engineering Research Council of Canada (NSERC)RGPIN-227233-2009 to K.M.
Data Polymorphism and the continuous variation of curvatures are two peculiar features of HIV capsids [2]. A HIV capsid protein consists of two independently structured domains, the N-terminus domain (NTD) and C-terminus domain (CTD), linked by a short flexible inter-domain linker [3]. HIV capsid proteins dimerize via helix 9 at its CTD in solution, and can form polymorphic assemblies in vitro[3]. Various structural models of such assemblies were determined, stabilized by three intermolecular contacts: NTD–NTD, NTD–CTD and trimeric interfaces [3]. We demonstrate the construction of a novel coarse grain (CG) model that captures the subtle variations of backbone structure of HIV capsid proteins and a strategy to account for protein dynamics with a static ensemble of subunits in conformations derived from all atom Molecular Dynamics (MD) simulations. Simulations using this novel CG model and strategy demonstrate that the variations of inter-domain motions controls the curvature of the assembly and causes the polymorphism, as show in Ref. [1]. In this article, we focus on the illustration of CG model conversion from the template pdb files and extraction of inter-domain motions from all atom MD simulation and solution NMR data. Fig. 1 illustrates the structural differences of the four experimental structural models of HIV capsid proteins utilized as templates for our novel coarse grain model: the isolated hexamer 3H47.pdb, tubular assembly 3J34.pdb, isolated pentamer 3P05.pdb and C-terminus dimer 2KOD.pdb. We show that our CG model uses cylinders to capture the subtle variations of backbone structures in these templates.