Folding And Assembly Of C-reactive Protein In Live Cells

As the main executor of life activities,protein functions entirely dependent on specific spatial structure.Mistakes in protein folding always induce diseases.Nevertheless,it remains unclear how protein's primary sequence determines its spatial structure.For proteins containing disulfide bond,the structure complexity is further increased.During the protein oxidative folding,the conformational folding and disulfide bonding may interfere with each other,theoretically making the folding process more difficult.To explore the co-ordination mechanisms between conformational folding and disulfide bonding in protein oxidative folding,we use C-reactive protein(CRP)as a case to study its folding mechanism in live cells.As an important acute phase protein in human body,CRP is a pentamer polymerized by five identical subunits in a no-covalent way.Each CRP subunit was equipped with a pair of intrasubunit disulfide bond.When the infections or inflammations happen,CRP expression will increase hundreds of times in a short period of time,indicating an efficient folding and assembly mechanism in vivo.Moreover,native CRP can be produced and secreted by E.coli with recombinant expression.In the present work,we expressed a series of CRP mutants with COS-7 and E.coli cells.Possible folding intermediates were captured and the folding orders of each secondary elements were carefully analysed,which allowed us to describe the exact folding and assembly mechanism in live cells.Our results reveal that CRP generates its pentameric structure with two sequencial steps.Firstly,CRP peptide folds into a near-native subunit.Then the assembly of pentamer occurs using the folded subunits.For the folding process of CRP subunits,it also contains two sequencial stages.At the early stage,strands C-I fold spontaneously into a hydrophobic core,companied with the correct position of a C-terminal helix.These together draw close the strands C&H and directly induce the formation of Cys36-Cys97 disulfide bond.By contrast,the second stage of CRP subunit folding involves extensive structure remolding and is not spontaneous.In this stage,the formed Cys36-Cys97 disulfide bond stabilizes the folded core and allows the strands J&K to integrate into the core in a calcium binding-guide way.Finally,the whole process of CRP subunit folding is completed.As a whole,we show that spontaneous conformational folding drives the formation of disulfide bond in the early stage of CRP subunit folding.In the subsequent folding process which is no-spontaneous,instead,the formed disulfide bond drives the conformational folding and assembly.Conclusions here thus clearly depicts a coordination relationship between conformation folding and disulfide bonding in the protein oxidative folding,which supplements and perfects the previous conclusions in this field to some extent.In addition,our results also argue that CRP generates its native structure following a specific pathway involving obligatory intermediates rather than undergoing numerous folding routes with random selections.