High Five! Methyl Probes at Five Ring Positions of Phenylalanine Explore the Hydrophobic Core Dynamics of Zinc Finger Miniproteins

Overview

Journal Chem Sci

Publisher Royal Society of Chemistry

Specialty Chemistry

Date 2021 Oct 20

PMID 34667551

Citations 2

Authors

Philip Horx

Armin Geyer

Affiliations

Soon will be listed here.

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 -, -, -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.

Citing Articles

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure.

Rua A, Alexandrescu A Protein Sci. 2024; 33(9):e5149.

PMID: 39180464 PMC: 11344264. DOI: 10.1002/pro.5149.

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure.

Rua A, Alexandrescu A bioRxiv. 2024; .

PMID: 38645208 PMC: 11030341. DOI: 10.1101/2024.04.06.588434.

References

Wuttke A, Geyer A . Self-assembly of peptide boroxoles on cis-dihydroxylated oligoamide templates in water. J Pept Sci. 2017; 23(7-8):549-555. DOI: 10.1002/psc.3007. View

Karplus M . Molecular dynamics simulations of biomolecules. Acc Chem Res. 2002; 35(6):321-3. DOI: 10.1021/ar020082r. View

Viles J, Patel S, Mitchell J, Moody C, Justice D, Uppenbrink J . Design, synthesis and structure of a zinc finger with an artificial beta-turn. J Mol Biol. 1998; 279(4):973-86. DOI: 10.1006/jmbi.1998.1764. View

Miao Y, Feher V, McCammon J . Gaussian Accelerated Molecular Dynamics: Unconstrained Enhanced Sampling and Free Energy Calculation. J Chem Theory Comput. 2015; 11(8):3584-3595. PMC: 4535365. DOI: 10.1021/acs.jctc.5b00436. View

Jha A, Saha S, Ayasolla K, Vashistha H, Malhotra A, Skorecki K . MiR193a Modulation and Podocyte Phenotype. Cells. 2020; 9(4). PMC: 7226544. DOI: 10.3390/cells9041004. View

Li W, Zhang J, Wang W . Understanding the folding and stability of a zinc finger-based full sequence design protein with replica exchange molecular dynamics simulations. Proteins. 2007; 67(2):338-49. DOI: 10.1002/prot.21312. View

Eckhardt B, Grosse W, Essen L, Geyer A . Structural characterization of a beta-turn mimic within a protein-protein interface. Proc Natl Acad Sci U S A. 2010; 107(43):18336-41. PMC: 2972999. DOI: 10.1073/pnas.1004187107. View

Gersbach C, Gaj T, Barbas 3rd C . Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies. Acc Chem Res. 2014; 47(8):2309-18. PMC: 4139171. DOI: 10.1021/ar500039w. View

Teilum K, Brath U, Lundstrom P, Akke M . Biosynthetic 13C labeling of aromatic side chains in proteins for NMR relaxation measurements. J Am Chem Soc. 2006; 128(8):2506-7. DOI: 10.1021/ja055660o. View

10.

Priem C, Wuttke A, Berditsch M, Ulrich A, Geyer A . Scaling the Amphiphilic Character and Antimicrobial Activity of Gramicidin S by Dihydroxylation or Ketal Formation. J Org Chem. 2017; 82(23):12366-12376. DOI: 10.1021/acs.joc.7b02177. View

11.

Kochoyan M, Keutmann H, Weiss M . Architectural rules of the zinc-finger motif: comparative two-dimensional NMR studies of native and "aromatic-swap" domains define a "weakly polar switch". Proc Natl Acad Sci U S A. 1991; 88(19):8455-9. PMC: 52527. DOI: 10.1073/pnas.88.19.8455. View

12.

Qian X, Weiss M . Two-dimensional NMR studies of the zinc finger motif: solution structures and dynamics of mutant ZFY domains containing aromatic substitutions in the hydrophobic core. Biochemistry. 1992; 31(33):7463-76. DOI: 10.1021/bi00148a006. View

13.

Weininger U, Respondek M, Low C, Akke M . Slow aromatic ring flips detected despite near-degenerate NMR frequencies of the exchanging nuclei. J Phys Chem B. 2013; 117(31):9241-7. DOI: 10.1021/jp4058065. View

14.

Baker E, Bartlett G, Porter Goff K, Woolfson D . Miniprotein Design: Past, Present, and Prospects. Acc Chem Res. 2017; 50(9):2085-2092. DOI: 10.1021/acs.accounts.7b00186. View

15.

Bulyk M, Huang X, Choo Y, Church G . Exploring the DNA-binding specificities of zinc fingers with DNA microarrays. Proc Natl Acad Sci U S A. 2001; 98(13):7158-63. PMC: 34639. DOI: 10.1073/pnas.111163698. View

16.

Igumenova T, Frederick K, Wand A . Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution. Chem Rev. 2006; 106(5):1672-99. PMC: 2547146. DOI: 10.1021/cr040422h. View

17.

Klevit R, Herriott J, Horvath S . Solution structure of a zinc finger domain of yeast ADR1. Proteins. 1990; 7(3):215-26. DOI: 10.1002/prot.340070303. View

18.

Lombardo C, Kumar M V V, Douat C, Rosu F, Mergny J, Salgado G . Design and Structure Determination of a Composite Zinc Finger Containing a Nonpeptide Foldamer Helical Domain. J Am Chem Soc. 2019; 141(6):2516-2525. DOI: 10.1021/jacs.8b12240. View

19.

Morita I, Mori T, Mitsuhashi T, Hoshino S, Taniguchi Y, Kikuchi T . Exploiting a C-N Bond Forming Cytochrome P450 Monooxygenase for C-S Bond Formation. Angew Chem Int Ed Engl. 2019; 59(10):3988-3993. DOI: 10.1002/anie.201916269. View

20.

Wuthrich K, Wagner G . NMR investigations of the dynamics of the aromatic amino acid residues in the basic pancreatic trypsin inhibitor. FEBS Lett. 1975; 50(2):265-8. DOI: 10.1016/0014-5793(75)80504-7. View