title: Characterization of the unfolding process of the tetrameric and dimeric forms of Cratylia mollis seed lectin (CRAMOLL 1): effects of natural fragmentation on protein stability.
abstract: pCRAMOLL 1 is a major, non-glycosylated isolectin found in seeds of Cratylia mollis, which belongs to the Leguminosae family and the Diocleinae subtribe. The lectin (~25 kDa) consists of 236 amino acids, sharing 82% identity and virtually identical topological architecture with concanavalin A. Both lectins also share the same pH-dependent dimer-tetramer equilibrium and the ability to recognize Glc/Man moieties. Intricate post-translational events occurring in Diocleinae seed cotyledons result in a mixture of intact and fragmented monomers within the oligomeric assemblies of pCRAMOLL 1. In an earlier report, we demonstrated the production, purification, and characterization of the bacterially expressed form of CRAMOLL 1 (rCRAMOLL 1). The recombinant lectin retained sugar-binding activity and several other biophysical properties of pCRAMOLL 1, but its tetramers, which are composed of intact monomers only, show little enhancement in stability when probed with acidification, high temperatures, or hydrostatic pressure. Here we examined the urea-induced unfolding of the nonfragmented tetramers and dimers of rCRAMOLL 1 and compared this behavior with that of the mixed plant lectin counterparts. Using fluorescence, circular dichroism, size-exclusion chromatography, and chemical cross-linking experiments, we posited that the absence of fragmentation lent greater firmness to tetramers, but not to dimers. Dimeric and tetrameric pCRAMOLL 1 unfolded via a compact monomeric intermediate. In contrast, dimers of rCRAMOLL 1 behaved similarly to the plant dimer counterpart, but its tetrameric form remarkably showed no evidence of such partially unfolded monomers. By analyzing the crystal structure of pCRAMOLL 1, we were able to dissect the importance of the fragmentation to lectin stability.
authors.affiliation: Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, 21941-902, Rio de Janeiro, RJ, Brazil.
authors.forename: Nathalia
authors.initials: N
authors.lastname: Varejão
authors.forename: Maria Tereza S
authors.initials: MT
authors.lastname: Correia
authors.forename: Debora
authors.initials: D
authors.lastname: Foguel
mesh_terms: D003432:Cross-Linking Reagents / Q000494:pharmacology; D007887:Fabaceae; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D037121:Plant Lectins / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding / Q000187:drug effects; D012639:Seeds; D014508:Urea / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D037121:Plant Lectins; D014508:Urea