Exploring the Structural Dynamics of the E. coli Chaperonin GroEL Using Translation-libration-screw Crystallographic Refinement of Intermediate States Charu Chaudhry 1 , Arthur L. Horwich 2 , Axel T. Brunger 3 and Paul D. Adams 4 * 1 Department of Molecular Biophysics and Biochemistry Yale University, New Haven CT 06520, USA 2 Department of Genetics and Howard Hughes Medical Institute, Yale University New Haven, CT 06510, USA 3 Department of Molecular and Cellular Physiology, Neurology and Neurological Sciences and Stanford Synchrotron Radiation Laboratory and Howard Hughes Medical Institute, Stanford University Stanford, CA 94305, USA 4 Lawrence Berkeley National Laboratory, One Cyclotron Road BLDG 4R0230, Berkeley, CA 94720-8235, USA Large rigid-body domain movements are critical to GroEL-mediated protein folding, especially apical domain elevation and twist associated with the formation of a folding chamber upon binding ATP and co- chaperonin GroES. Here, we have modeled the anisotropic displacements of GroEL domains from various crystallized states, unliganded GroEL, ATPgS-bound, ADP-AlFx/GroES-bound, and ADP/GroES bound, using translation-libration-screw (TLS) analysis. Remarkably, the TLS results show that the inherent motions of unliganded GroEL, a polypeptide- accepting state, are biased along the transition pathway that leads to the folding-active state. In the ADP-AlFx/GroES-bound folding-active state the dynamic modes of the apical domains become reoriented and coupled to the motions of bound GroES. The ADP/GroES complex exhibits these same motions, but they are increased in magnitude, potentially reflecting the decreased stability of the complex after nucleotide hydrolysis. Our results have allowed the visualization of the anisotropic molecular motions that link the static conformations previously observed by X-ray crystal- lography. Application of the same analyses to other macromolecules where rigid body motions occur may give insight into the large scale dynamics critical for function and thus has the potential to extend our fundamental understanding of molecular machines. Published by Elsevier Ltd. Keywords: chaperonin; translation-libration-screw; crystallographic refine- ment; structural dynamics; protein folding *Corresponding author Introduction The molecular chaperonin GroEL, along with its co-chaperonin GroES, form a dynamic macromol- ecular complex that mediates protein folding in the bacterial cell, concurrent with the consumption of ATP. 1 GroEL is made of two heptameric rings that stack back-to-back with dyad symmetry. 2 Non- native polypeptides bind to hydrophobic sites located on the apical domains at the end of the central channel 3–7 (Figure 1(i) and (v)). Folding is initiated by the positive cooperative binding of seven ATP molecules 8–11 followed by the binding of the heptameric co-chaperonin GroES 12 to the same ring (cis ring). This generates large, nearly rigid body movement of the intermediate and apical domains of the cis-ring subunits that dramatically enlarges and seals the GroEL cis cavity. The process of forming this sealed chamber, referred to as the Anfinsen cage, occludes the hydrophobic binding surfaces, thereby releasing the polypeptide into the cavity to initiate folding 13–15 (Figure 1(ii) and (iii)). The GroEL–GroES complex holds the polypeptide for wten seconds in a hydrophilically lined cav- ity. 16–19 Following ATP hydrolysis (Figure 1(iv)), the products of this “half-cycle”, ADP and product polypeptide, are released along with GroES, as another similar folding-active assembly is formed in the opposing or trans ring 19,20 (Figure 1(v) and (vi)). The behavior of the trans ring is 1808 out of phase with the cis ring, due to the negative cooperativity between the rings. 21,22 Several conformational states of the GroEL–GroES 0022-2836/$ - see front matter Published by Elsevier Ltd. Abbreviations used: ADPs, anisotropic displacement parameters; TLS, translation-libration-screw; FOM, figures-of-merit; NCS, non-crystallographic symmetry. E-mail address of the corresponding author: pdadams@lbl.gov DTD 5 ARTICLE IN PRESS doi:10.1016/j.jmb.2004.07.015 J. Mol. Biol. (2004) xx, 1–17