Protein Structure,Function And Stability In Complex Evironment Studied By NMR

The cell environment is extremely complex where macromolecules,spatial confinement and weak interaction are ubiquitous.The natural cell environment where proteins function may affect protein's structure,function and stability.Nuclear Magnetic Resonance(NMR)can provide information about protein's structure,function and stability at atomic resolution.NMR is capable of studying the infulence of environment effects such as crowding and confinement on protein's structure in solution state.We used NMR to study how the confined environment of reverse micelles(RMs)on multidomain protein's sturcture and function.We utilized 19F NMR methods to study how crowding and confinement affect KH1 stability.The confined environment of RMs affects multidomain protein's structureProtein confinement is not only a key aspect of many biological processes,including folding and misfolding in chaperones,protein biosynthesis in ribosome tunnel,and protein transport in translocons,but also plays important roles in industrial applications,such as drug delivery,enzyme retrieval,and therapeutic product protection.Little is known about how confinement affects the structure of multidomain proteins.We utilized NMR methods to study how confined environment of RMs affect the structures of multidomain protein calmodulin and its complex.Our results showed that confinement didn't affect the structure of mutlidomain protein complex holoCaM-MLCK,which forms compact rigid structure in dilute solution.HoloCaM forms an extended structrue in dilute solution.Confinement forces holoCaM to form a compact structure with interdomain contacts.Except where the orientation of the fourth helix in both domains shows some differences,the overall structures of the N-terminal and C-terminal domains are similar to those of the extended structureThe confined environments of RMs alter holoCaM functionWe carried out 1H-15N HSQC titration experiments on HoloCaM to investigate whether the structural change induced by confinement affects function.Our results show confinement change the function of CaM.CaM binds the MLCK,AcN19 and somatostatin peptides in dilute buffer,but only binds the MLCK and AcN19 in reverse micelles.In dilute buffer,the binding of MLCK and AcN19 peptides to holoCaM results in a compact structure,respectively,although the binding affinity is remarkably different.The PCSs and RDCs data on the CaM-somatostatin complex suggest that the complex exists mainly in an extended form.The structural difference implies that CaM binding MLCK and AcN 19,but not somatostatin,in RMs is likely the result of volume selection,rather than binding affinity.The extended structure of the CaM-somatostatin complex takes larger space compared to the compact structure.Owing to the limited space in RMs,CaM selects to form a compact structure by itself,rather than interact with the somatostatin peptide.Thermodynamics of protein folding and binding in complex environmentsThe folding and binding of proteins in cell often occur in crowded and confined environment.Studies have shown crowding and confinement can change proteins'stabilities.However few studies have been performed to systematically analyse the stabilities of proteins in complex environment.We utilized 19F NMR to study the effects of crowding and confinement on protein stability.We found that KH1 is stabilized in crowding agents,which is entropic stabilized and enthalpic destabilized.The monomers(sucrose,EG,glucose)are more stabilizing than the polymers(ficoll 70,PEG 200,dextran 70).KH1 is more entropically stabilized and enthalpically destabilized in monomers than in polymers,which is against the theory of macromolecular crowding.KH1 is destabilized in RMs,which are enthalpically stabilized and entropically destabilized.Our results are in sharp contrast with predictions of the hard-core excluded volume theory.The chemical interactions between the group on the interior of RMs and the test protein probably work.The geometry of the chemical interactions may aslo play a role.In summary,we use NMR to investigate the effects of complex environment on the structure,function and stability of proteins.We believe that the cells may use the environment effects to regulate the structrue,function and stability of proteins.