High five! Methyl probes at five ring positions of phenylalanine explore the hydrophobic core dynamics of zinc finger miniproteins.

Philip Horx,Armin Geyer

doi:10.1039/d1sc02346b

Abstract

The elucidation of internal dynamics in proteins is essential for the understanding of their stability and functionality. Breaking the symmetry of the degenerate rotation of the phenyl side chain provides additional structural information and allows a detailed description of the dynamics. Based on this concept, we propose a combination of synthetic and computational methods, to study the rotational mobility of the Phe ring in a sensitive zinc finger motif. The systematic methyl hopping around the phenylalanine ring yields o-, m-, p-tolyl and xylyl side chains that provide a vast array of additional NOE contacts, allowing the precise determination of the orientation of the aromatic ring. MD simulations and metadynamics complement these findings and facilitate the generation of free energy profiles for each derivative. Previous studies used a wide temperature window in combination with NMR spectroscopy to elucidate the side chain mobility of stable proteins. The zinc finger moiety exhibits a limited thermodynamic stability in a temperature range of only 40 K, making this approach impractical for this compound class. Therefore, we have developed a method that can be applied even to thermolabile systems and facilitates the detailed investigation of protein dynamics.

Highlights

Side chain rotations make protein structures dynamic so they can ful ll their functional duties like molecular recognition, catalysis, and signal transmission to name a few examples
Only the lowest NMR structure for each variant is depicted in Fig. 5, while an ensemble of 10 conformers is represented in the Electronic supplementary information (ESI).† Generally, all derivatives were able to adopt a zinc nger type fold, as typical chemical shi values remained the same and TOCSY/ NOESY patterns differentiated only slightly
The results indicate that the hydrophobic core opposes certain geometrical restrictions

Summary

Introduction

Side chain rotations make protein structures dynamic so they can ful ll their functional duties like molecular recognition, catalysis, and signal transmission to name a few examples. While the general idea of studying dynamics by applying temperature gradients is still used today, its application is limited to only the most stable proteins and hampered by the number of aromatic NMR signals.[5] A further development is the use of relaxation dispersion experiments which provided excellent agreement with simulations[6] and allows the study of near degenerate spin systems.[7,8,9,10] Here, we propose a method using phenylalanine derivatives with modi ed side chains in combination with molecular dynamics simulation, to characterize the hydrophobic core dynamics in miniproteins with limited thermodynamic stability.

Results

Conclusion