Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

Xiancheng Zeng,Huan-Xiang Zhou,Vann Bennett,Whasil Lee,Piotr E. Marszalek,Weitao Yang

doi:10.1074/jbc.m110.179697

Abstract

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and is [corrected] under severe conformational constraints [corrected] dictated by the one-dimensional geometry of the tunnel. [corrected] How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.

Highlights

Sis, the nascent polypeptide chain (NPC)4 is extruded through the long (ϳ80 Å) and narrow (10 –20 Å) ribosome exit tunnel in which the NPC starts its folding process [40]
It was chosen as our model system because of (i) the fact that ankyrin repeat (AR) are very common and have been identified in over 4700 proteins [25]; (ii) its extended “vectorial” structure; (iii) its composition consisting of tandem repeats of nearly identical sequences, which should simplify the analysis of force spectroscopy data; (iv) expected robust refolding forces that can be captured by atomic force microscope (AFM) [67,68,69]; and (v) its extreme thermodynamic stability [66] that makes mechanical stretching and relaxing the only practical experimental approach to induce and follow the repeats’ unfolding and refolding
The steered molecular dynamics (SMD) trace was shifted to the right by 20 nm to compensate for the initial length of I27 modules that contribute to the extension in the AFM measurements but are absent in the SMD simulations

Summary

Introduction

Sis, the nascent polypeptide chain (NPC) is extruded through the long (ϳ80 Å) and narrow (10 –20 Å) ribosome exit tunnel in which the NPC starts its folding process [40]. As recently suggested by Cabrita et al [41], the vectorial character of co-translational folding is in a way mimicked by force-induced unfolding experiments Such mechanical experiments can be carried out, for example, in an atomic force microscope (AFM) (48 –54), with optical tweezers [10,11], or by translocating proteins through a pore [55,56,57]. It was chosen as our model system because of (i) the fact that ARs are very common and have been identified in over 4700 proteins [25]; (ii) its extended “vectorial” structure; (iii) its composition consisting of tandem repeats of nearly identical sequences, which should simplify the analysis of force spectroscopy data; (iv) expected robust refolding forces that can be captured by AFM [67,68,69]; and (v) its extreme thermodynamic stability [66] that makes mechanical stretching and relaxing the only practical experimental approach to induce and follow the repeats’ unfolding and refolding

Methods

Results

Conclusion