Folding Coupled with Assembly in Split Green Fluorescent Proteins

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Folding Coupled with Assembly in Split Green Fluorescent Proteins Studied by Structure-Based Molecular Simulations Mashiho Ito, Takeaki Ozawa, and Shoji Takada J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp4032817 • Publication Date (Web): 16 May 2013 Downloaded from http://pubs.acs.org on May 20, 2013

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The Journal of Physical Chemistry

Folding Coupled with Assembly in Split Green Fluorescent Proteins Studied by Structure-based Molecular Simulations Mashiho Ito, Takeaki Ozawa∗, and Shoji Takada† May 16, 2013

Department of Chemistry, School of Science, The University of Tokyo, Department of Biophysics, Graduate School of Science, Kyoto University

Abstract Split green fluorescent protein (GFP) is a powerful tool for imaging of protein-protein interactions in living cells, but molecular mechanisms of the folding and the assembly of split GFPs are poorly understood. Here, using a simple Go model that is based on the energy landscape theory, we performed comprehensive folding simulations of six split GFPs with different split points. Of the six, the fluorescence recovery was reported in four, but not in the other two. In the simulations, we found that, when ∗ [email protected]

† [email protected]

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ACS Paragon Plus Environment


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the complete folding and assembly were observed, the N-terminal fragment always folded earlier than the C-terminal fragment. The in silico folding rates of the split GFPs were larger for the four split GFPs that the fluorescence recovery was reported in literature. The stability of standalone N-terminal fragments were well correlated with the folding rates of split GFPs. These suggest that the efficient folding and assembly of split GFPs are realized when the N-terminal fragment folds spontaneously with the central α-helix as a nucleation core and that the C-terminal fragment folding is coupled with the assembly to the pre-formed N-terminal fragment.

Keywords: imaging, GFP, energy landscape theory, coarse-grained, Go model, CafeMol

Introduction Green fluorescent protein (GFP) and its variants are now widely used to examine spatiotemporal patterns and protein interactions in living cells. Particularly, protein-protein interactions and their subcellular localization in cells and organisms can be identified by complementation or reconstitution of split GFP fragments.[1, 2, 3, 4, 5] In this method, GFP is cleaved into two fragments, Nand C-terminal fragments, which are genetically connected to proteins “A” and “B”, respectively (Fig. 1). When the protein A interacts with the protein B, two fragments of the split GFP assemble and fold into the three-dimensional structure of GFP, leading to the fluorescence recovery. The reassembly of the

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GFP is an essentially irreversible process, and the minimum affinity required for the reassembly is 500 µM to 1 mM .[6] Such special character provides applicability of the analysis of different pairs of protein-protein interactions, but molecular mechanisms of the folding and the assembly process are unclear. GFP takes a β-barrel topology made of 11 β-strands, which encircles one central α-helix. After the completion of the β-barrel folding, a self-catalyzed reaction takes place at three residues in the central α-helix that form the chromophore. It is known that the complete folding of GFP is a prerequisite for the chromophore formation and the fluorescence activity. Thus, it is crucial to understand folding mechanisms of GFP. Indeed, folding reactions of GFP and its variants have been investigated both experimentally [7, 8] and theoretically .[9, 10, 11, 12] Comparing to GFPs, the complete folding of split GFPs involves assembly of the two fragments, which makes the fluorescence recovery process more complex. Currently, molecular mechanisms of assembly and folding of split GFPs are poorly understood. There are many questions to be addressed. First, through intensive studies, the fluorescence recovery is reported for many split points, which are mostly located in certain loops, but not in other loops. Why are some split points successful, but not others? What are the conformational characteristics of individual fragments for various split points? Are these conformational properties of fragments related to the successful fluorescence recovery? The purpose of this paper is to address these questions by performing folding simulations based on the energy landscape theory of proteins.

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The energy landscape theory for protein folding was originated from the seminal paper by Bryngelson and Wolynes in 1987.[13] In their paper, foldability of proteins was first quantified by the ratio of the folding transition temperature to the glass transition temperature, which led to the principle of minimum frustration. Later, the principle was more pictorially represented by the, now very famous, folding funnel. [14, 15] The native state corresponds to the bottom of the funnel. The theory suggests that, as the conformation deviates from the native, on average the effective energy tends to increase. The perfect funnel can be realized by the so-called Go model [16, 17, 18], which, albeit very simple, turned out to be able to predict various aspects of protein folding reactions. [16, 19] The energy landscape theory was further applied to many other conformational dynamics [20, 21], such as the folding coupled with binding. [22, 23] Here, based on these studies, we apply Go model to the folding of split GFPs. The paper is organized in the following way. We start with the brief description of the Method. Then, in the Result section, we report folding simulations of split GFPs with various split points, finding that the folding rate is clearly dependent on the split point. To further address reasons of these diversity, we next conducted equilibrium sampling simulations of individual fragments. We found that the C-terminal fragment is almost always unfolded and that the stability of N-terminal fragments is well-correlated with the folding rate of split GFPs. These together led to a model for the split GFP folding, which is explained in the Discussions and Conclusion.

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Full-Length GFP Fluorescent C-term

N-term

N-term

"Split Point"

C-term

Fluorescent

A

B

Interaction of A with B

Complemen

-tation

B

A

A

B

Figure 1: Split GFP complementation. The full-length GFP is cleaved into N- and C-terminal fragments, which are genetically linked to proteins A and B, respectively. When proteins A and B interact each other, two fragments of GFP assemble and fold to the complete GFP form which recovers fluorescence. 193/194

155/156

39/40 4

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225/226

158/159

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10 11

6 1 2

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3 9

102/103 145/146 169/170

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173/174

Figure 2: Split points reported to recover fluorescence. Split GFP fragments with the split points at the red marked points are known to recover the fluorescence. For example, 158/159 means that GFP sequence is cleaved between 158 and 159. Numbers in squares indicate β-strand numbering.[24]

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Methods Sequence and structure of GFP studied For folding simulations with a Go model we need native structural data. Starting from the native structure of the enhanced-GFP (EGFP), (the protein data bank ID: 2Y0G), we removed the His-tag, and replaced the chromophore with their original triplet of amino acids. The side-chains of these amino acids, as well as some missing residues in N- and C- termini were modeled by Modeller.[25] This structure was used for the reference of the intact EGFP folding simulations. For split GFPs, the structure is divided into two fragments at different split points.

Folding simulation of intact GFP, and split GFP We used the Go model of Clementi et al for all simulations. [16] First, we used one CG particle per amino acid, which is located at the Cα position. Specifically, the energy function is

V ({ri })

=

∑

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∑

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i

∑ + {Kφ(1) [1 − cos(φi − φi,0 )] + Kφ(3) [1 − cos 3(φi − φi,0 )]} i

+

nat ∑ contact i

Folding Coupled with Assembly in Split Green Fluorescent Proteins

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