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Optimizing 19F NMR protein spectroscopy by fractional biosynthetic labeling

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Abstract

In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, 19F and 1H-15N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling. The effects of varying levels of enrichment of 3-fluorophenylalanine (i.e. between 3 and >95%) were further studied by 19F and 1H-15N HSQC spectra,15N T1 and T2 relaxation measurements, 19F T2 relaxation, translational diffusion and heat denaturation experiments via circular dichroism. Our results show that while several properties, including translational diffusion and thermal stability show little variation between non-fluorinated and >95% 19F labeled samples, 19F and 1H-15N HSQC spectra show significant improvements in line widths and resolution at or below 76% enrichment. Moreover, high levels of fluorination (>80%) appear to increase protein disorder as evidenced by backbone 15N dynamics. In this study, reasonable signal to noise can be achieved between 60–76% 19F enrichment, without any detectable perturbations from labeling.

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Abbreviations

3-FPhe:

3-fluorophenylalanine

CaM:

Calmodulin

NMR:

Nuclear magnetic resonance

References

  • Akcay G, Kumar K (2009) A new paradigm for protein design and biological self-assembly. J Fluor Chem 130:1178–1182

    Article  Google Scholar 

  • Altieri AS, Hinton DP, Byrd RA (1995) Association of biomolecular systems via pulsed-field gradient Nmr self-diffusion measurements. J Am Chem Soc 117:7566–7567

    Article  Google Scholar 

  • Anderluh G, Razpotnik A, Podlesek Z, Macek P, Separovic F, Norton RS (2005) Interaction of the eukaryotic pore-forming cytolysin equinatoxin ii with model membranes: F-19 Nmr studies. J Mol Biol 347:27–39

    Article  Google Scholar 

  • Bann JG, Pinkner J, Hultgren SJ, Frieden C (2002) Real-time and equilibrium F-19-Nmr studies reveal the role of domain-domain interactions in the folding of the chaperone papd. Proc Natl Acad Sci USA 99:709–714

    Article  ADS  Google Scholar 

  • Barbato G, Ikura M, Kay LE, Pastor RW, Bax A (1992) Backbone dynamics of calmodulin studied by N-15 relaxation using inverse detected 2-dimensional Nmr-spectroscopy-the central Helix is flexible. Biochemistry 31:5269–5278

    Article  Google Scholar 

  • Bilgiçer B, Fichera A, Kumar K (2001) A coiled coil with a fluorous core. J Am Chem Soc 123:4393–4399

    Article  Google Scholar 

  • Broos J, Maddalena F, Hesp BH (2004) In vivo synthesized proteins with monoexponential fluorescence decay kinetics. J Am Chem Soc 126:22–23

    Article  Google Scholar 

  • Campos-Olivas R, Aziz R, Helms GL, Evans JNS, Gronenborn AM (2002) Placement of F-19 into the center of Gb1: effects on structure and stability. FEBS Lett 517:55–60

    Article  Google Scholar 

  • Crivici A, Ikura M (1995) Molecular and structural basis of target recognition by calmodulin. Annu Rev Biophys Biomol Struct 24:85–116

    Article  Google Scholar 

  • Danielson MA, Falke JJ (1996) Use of F-19 Nmr to probe protein structure and conformational changes. Annu Rev Biophys Biomol Struct 25:163–195

    Article  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) Nmrpipe-a multidimensional spectral processing system based on unix pipes. J Biomol NMR 6:277–293

    Article  Google Scholar 

  • Duewel H, Daub E, Robinson V, Honek J (2001) Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein. Biochemistry 40:13167–13176

    Article  Google Scholar 

  • Geddes CD (2009) Reviews in Fluorescence 2008, vol 5, illustrated ed. Springer

  • Hoeflich KP, Ikura M (2002) Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108:739–742

    Article  Google Scholar 

  • Holz M, Weingartner H (1991) Calibration in accurate spin-echo self-diffusion measurements using H-1 and less-common nuclei. J Magn Reson 92:115–125

    Google Scholar 

  • Jarymowycz VA, Stone MJ (2006) Fast time scale dynamics of protein backbones: Nmr relaxation methods, applications, and functional consequences. Chem Rev 106:1624–1671

    Article  Google Scholar 

  • Johnson BA, Blevins RA (1994) Nmr view-a computer-program for the visualization and analysis of Nmr data. J Biomol NMR 4:603–614

    Article  Google Scholar 

  • Kay LE, Torchia DA, Bax A (1989) Backbone dynamics of proteins as studied by N-15 inverse detected heteronuclear Nmr-spectroscopy-application to staphylococcal nuclease. Biochemistry 28:8972–8979

    Article  Google Scholar 

  • Kitevski-LeBlanc JL, Evanics F, Prosser RS (2009) Approaches for the measurement of solvent exposure in proteins by F-19 Nmr. J Biomol NMR 45:255–264

    Article  Google Scholar 

  • Kitevski-Leblanc JL, Evanics F, Scott Prosser R (2010) Approaches to the assignment of (19)F resonances from 3-fluorophenylalanine labeled calmodulin using solution state NMR. J Biomol NMR 47:113–123

    Article  Google Scholar 

  • Li HL, Frieden C (2007) Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and F-19 Nmr. Proc Natl Acad Sci USA 104:11993–11998

    Article  ADS  Google Scholar 

  • Luck LA, Falke JJ (1991) F-19 Nmr-studies of the D-galactose chemosensory receptor.1. Sugar binding yields a global structural-change. Biochemistry 30:4248–4256

    Article  Google Scholar 

  • Masino L, Martin SR, Bayley PM (2000) Ligand binding and thermodynamic stability of a multidomain protein, calmodulin. Protein Sci 9:1519–1529

    Article  Google Scholar 

  • Nicholson LK, Kay LE, Baldisseri DM, Arango J, Young PE, Bax A, Torchia DA (1992) Dynamics of methyl-groups in proteins as studied by proton-detected C-13 Nmr-spectroscopy-application to the leucine residues of staphylococcal nuclease. Biochemistry 31:5253–5263

    Article  Google Scholar 

  • Okano H, Cyert MS, Ohya Y (1998) Importance of phenylalanine residues of yeast calmodulin for target binding and activations. J Biol Chem 273:26375–26382

    Article  Google Scholar 

  • Quint P, Ayala I, Busby SA, Chalmers MJ, Griffin PR, Rocca J, Nick HS, Silverman DN (2006) Structural mobility in human manganese superoxide dismutase. Biochemistry 45:8209–8215

    Article  Google Scholar 

  • Rajagopalan S, Chow C, Raghunathan V, Fry C, Cavagnero S (2004) Nmr spectroscopic filtration of polypeptides and proteins in complex mixtures. J Biomol NMR 29:505–516

    Article  Google Scholar 

  • Rozovsky S, Jogl G, Tong L, McDermott AE (2001) Solution-state Nmr investigations of triosephosphate isomerase active site loop motion: ligand release in relation to active site loop dynamics. J Mol Biol 310:271–280

    Article  Google Scholar 

  • Schuler B, Kremer W, Kalbitzer HR, Jaenicke R (2002) Role of entropy in protein thermostability: folding kinetics of a hyperthermophilic cold shock protein at high temperatures using F-19 Nmr. Biochemistry 41:11670–11680

    Article  Google Scholar 

  • Tang Y, Ghirlanda G, Petka W, Nakajima T, DeGrado W, Tirrell D (2001) Fluorinated Coiled-coil proteins prepared in vivo display enhanced thermal and chemical stability. Angew Chem Int Ed 40:1494–1496

    Article  Google Scholar 

  • Visser NV, Westphal AH, Nabuurs SM, van Hoek A, van Mierlo CPM, Visser A, Broos J, van Amerongen H (2009) 5-Fluorotryptophan as dual probe for ground-state heterogeneity and excited-state dynamics in apoflavodoxin. FEBS Lett 583:2785–2788

    Article  Google Scholar 

  • Weljie A, Yamniuk A, Yoshino H, Izumi Y, Vogel H (2003) Protein conformational changes studied by diffusion Nmr spectroscopy: application to helix-loop-helix calcium binding proteins. Protein Sci 12:228–236

    Article  Google Scholar 

  • Woll MG, Hadley EB, Mecozzi S, Gellman SH (2006) Stabilizing and destabilizing effects of phenylalanine ->F-5-phenylalanine mutations on the folding of a small protein. J Am Chem Soc 128:15932–15933

    Article  Google Scholar 

  • Woolfson DN (2001) Core-directed protein design. Curr Opin Struct Biol 11:464–471

    Article  Google Scholar 

  • Xiao GY, Parsons JF, Tesh K, Armstrong RN, Gilliland GL (1998) Conformational changes in the crystal structure of rat glutathione transferase M1–1 with global substitution of 3-fluorotyrosine for tyrosine. J Mol Biol 281:323–339

    Article  Google Scholar 

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Acknowledgments

We are grateful to Prof. Mitsu Ikura (University of Toronto) for providing a plasmid for Xenopous laevis CaM used in this study and to Prof. Avi Chakrabartty (University of Toronto) for the use of the CD apparatus. We thank Sameer Al-Abdul Wahid (University of Toronto at Mississauga) for assistance with the diffusion experiments. JK-L wishes to acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for a doctoral fellowship. RSP acknowledges NSERC, for financial support through the NSERC discovery grant program (261980) and CIHR for support through a Protein Folding Training Grant.

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Authors and Affiliations

  1. Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd., North Mississauga, ON, L5L 1C6, Canada

    Julianne L. Kitevski-LeBlanc, Ferenc Evanics & R. Scott Prosser

  2. Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada

    R. Scott Prosser

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  1. Julianne L. Kitevski-LeBlanc
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  2. Ferenc Evanics
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  3. R. Scott Prosser
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Correspondence to R. Scott Prosser.

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Kitevski-LeBlanc, J.L., Evanics, F. & Scott Prosser, R. Optimizing 19F NMR protein spectroscopy by fractional biosynthetic labeling. J Biomol NMR 48, 113–121 (2010). https://doi.org/10.1007/s10858-010-9443-7

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