Structural Characterization of Dynamic Protein Machines by Cryo-electron Microscopy

Electron Microscopy;Chaperones;Biological Chemistry;Science;Chemical Biology

Yokom, AdamTrievel, Raymond C ;

University of Michigan

关键词: Electron Microscopy;    Chaperones;    Biological Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/140811/alyokom_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Conformational changes of proteins are a primary factor for proper cellular signaling, protein folding and substrate interaction. Specifically, molecular chaperones, which are required to maintain protein homeostasis, traverse large conformational landscapes to bind client proteins. However, studying the conformational changes in these proteins has long been a challenge. Recent advancements in electron microscopy (EM) have dramatically improved our ability to capture protein conformational states. Therefore, EM was utilized as a structural tool to further our understanding of two dynamic protein systems, Heat shock protein 104 (Hsp104) and nitric oxide synthase(NOS). Yeast Hsp104 is a powerful disaggregase that functions to pull apart amorphous and amyloid aggregates. Hsp104 contains two AAA+ domains which, through a mechanism not fully understood, translocates substrate through its central channel. Cryo EM 3D reconstructions of three unique Hsp104 conformations have been determined. Hsp104-AMPPNP was observed in an open state (5.6Å) and shows a large asymmetric offset around the AAA+ domains. This spiral arrangement of the AAA+ domains creates a unique heteromeric NBD1-NBD2 interaction. Furthermore, the open state captured structural information on many critical regulatory domains, such as the middle domain, N-terminal domain and C-terminal domain. 3D classification of a substrate bound Hsp104-ATPS complex revealed a mix of closed and extended states to ~4Å resolution. Tyrosine pore loop contacts are seen to interact directly with the polypeptide backbone threaded into the central channel of the Hsp104 hexamer. Moreover, these structures revealed a two-residue translocation step for substrate threading in Hsp104. Together these high-resolution reconstructions have led to a novel model of Hsp104 function, which encompasses both nonprocessive and processive substrate translocation.Secondly, EM techniques were used to study Nitric Oxide Synthase (NOS). NOS is a critical signaling protein and is the sole source of NO within mammals. NOS involves a complex electron transfer cycle to convert L-arginine to NO and L-citrulline. This vital enzyme has been a challenge to study structurally, and full-length information has been lacking. By utilizing 2D and 3D classification methods we were able to obtain the first full-length structure of nNOS bound to its critical regulatory partner, calmodulin (CaM). 2D analysis revealed a large degree of flexibility around the NOS dimer showing both open, intermediate and closed states. By using glutaraldehyde crosslinking and random conical tilt, a 23Å reconstruction of nNOS-CaM showed how CaM binding allows FMN ;;deshielding’. The FMN domain undergoes a 115° rotation away from the flavin core, and toward the heme co-factor allowing for efficient electron transfer and subsequent NO production. Mechanistic, structural and functional information have been determined for both Hsp104 and NOS and the details of these studies follow.