Investigation Of The Structure And Dynamic Of Im7 Protein In Natural Outer Membrane Vesicles Via NMR

Functions of molecules,in many cases,is highly dependent on their conformations and hence intimately coupled to the structure,dynamics,and flexibility.For biomacromolecules,such as proteins,interactions between sidechains of proteins within themself and with the surrounding environments determine the final folding status of proteins.Thus,the environment surrounding proteins is critical for their structures and can dramatically affect the conformational equilibria and subsequently activities of proteins.Nuclear magnetic resonance(NMR)spectroscopy is a powerful tool for studying the structure of molecules,especially for in-situ conformations,and dynamics.Nevertheless,the limited lifetime of living cells inside the NMR tube and the low concentrations of target proteins in cells hinder the acquisition of experiments longer than a few hours via the in-cell NMR technique.Thus,the development of new in-situ NMR system to investigate protein structures,and dynamics is urgent.In this dissertation,new systems and methods are developed to address the aforementioned two problems:1.Establishment of a new system for in situ NMR investigations of protein structures and dynamicsThe outer member vesicles(OMVs)are spherical extracellular vesicles released by cells in a controlled manner.Similar to the cellular environment,the environment of the OMVs is complicated,containing various metabolites.DNAs,RNAs,and crowding proteins.The concentration of all bio-logical contents closely resembles the periplasmic space of bacterial cells with a quasi-cellular context,exceeding 300 mg/mL.However,the inanimate properties of OMVs enable long-time NMR measurements,providing a way for further in-situ investigation of protein dynamics with this vesicle-based solution NMR spectroscopy.2.Investigation of the conformational exchanges of Im7 in E.coli OMVsWe revealed the immunity protein Im7 was less frustrated and shifted toward its ground state in OMVs than in the aqueous solution.Further experiments showed both macromolecular crowding and quinary interactions with the periplasmic components stabilized the ground state of Im7.Our studies highlight the key role of the OMVs environment played on protein conformational equilibria and subsequently conformation-related protein functions.3.Investigation of the chaperone roles of HdeA and Spy in OMVsHigh resolution NMR spectra of HdeA in OMVs were obtained.The results revealed the conformational changes of HdeA in OMVs into its active state,further expanding the applications of OMVs in the investigation of periplasmic chaperone’s structures and dynamics in situ via nuclear magnetic spectroscopy.Moreover,the coexpression of the chaperone Spy and the client Im7 led to the enrichment of the SpyIm7 complex in OMVs,facilitating the study of the chaperone-client protein interaction in its native environment.The data showed both the N-terminal and C-terminal of Spy involved in its interaction with Im7 in OMVs.