Isotope-Labeled Amyloids Via Synthesis, Expression, and Chemical Ligation for Use in FTIR, 2D IR, and NMR Studies

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2015 Oct 11

PMID 26453203

Citations 3

Authors

Tianqi O Zhang

Maksim Grechko

Sean D Moran

Martin T Zanni

Affiliations

Soon will be listed here.

Abstract

This chapter provides protocols for isotope-labeling the human islet amyloid polypeptide (hIAPP or amylin) involved in type II diabetes and γD-crystallin involved in cataract formation. Because isotope labeling improves the structural resolution, these protocols are useful for experiments using Fourier transform infrared (FTIR), two-dimensional infrared (2D IR), and NMR spectroscopies. Our research group specializes in using 2D IR spectroscopy and isotope labeling. 2D IR spectroscopy provides structural information by measuring solvation from 2D diagonal lineshapes and vibrational couplings from cross peaks. Infrared spectroscopy can be used to study kinetics, membrane proteins, and aggregated proteins. Isotope labeling provides greater certainty in the spectral assignment, which enables new structural insights that are difficult to obtain with other methods. For amylin, we provide a protocol for (13)C/(18)O labeling backbone carbonyls at one or more desired amino acids in order to obtain residue-specific structural resolution. We also provide a protocol for expressing and purifying amylin from E. coli, which enables uniform (13)C or (13)C/(15)N labeling. Uniform labeling is useful for measuring the monomer infrared spectrum in an amyloid oligomer or fiber as well as amyloid protein bound to another polypeptide or protein, such as a chaperone or an inhibitor. In addition, our expression protocol results in 2-2.5 mg of amylin peptide per 1 L cell culture, which is a high enough yield to straightforwardly obtain the 2-10 mg needed for high resolution and solid-state NMR experiments. Finally, we provide a protocol to isotope-label either of the two domains of γD-crystallin using expressed protein ligation. Domain labeling makes it possible to resolve the structures of the two halves of the protein in FTIR and 2D IR spectra. With modifications, these strategies and protocols for isotope labeling can be applied to other amyloid polypeptides and proteins.

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References

Chung H, Khalil M, Smith A, Tokmakoff A . Transient two-dimensional IR spectrometer for probing nanosecond temperature-jump kinetics. Rev Sci Instrum. 2007; 78(6):063101. DOI: 10.1063/1.2743168. View

Dunkelberger E, Buchanan L, Marek P, Cao P, Raleigh D, Zanni M . Deamidation accelerates amyloid formation and alters amylin fiber structure. J Am Chem Soc. 2012; 134(30):12658-67. PMC: 3410046. DOI: 10.1021/ja3039486. View

Middleton C, Woys A, Mukherjee S, Zanni M . Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy. Methods. 2010; 52(1):12-22. PMC: 2933966. DOI: 10.1016/j.ymeth.2010.05.002. View

Kosicka I, Kristensen T, Bjerring M, Thomsen K, Scavenius C, Enghild J . Preparation of uniformly (13)C,(15)N-labeled recombinant human amylin for solid-state NMR investigation. Protein Expr Purif. 2014; 99:119-30. DOI: 10.1016/j.pep.2014.04.002. View

Flavell R, Muir T . Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biology. Acc Chem Res. 2008; 42(1):107-16. DOI: 10.1021/ar800129c. View

Falvo C, Zhuang W, Kim Y, Axelsen P, Hochstrasser R, Mukamel S . Frequency distribution of the amide-I vibration sorted by residues in amyloid fibrils revealed by 2D-IR measurements and simulations. J Phys Chem B. 2012; 116(10):3322-30. PMC: 3314731. DOI: 10.1021/jp2096423. View

Shim S, Gupta R, Ling Y, Strasfeld D, Raleigh D, Zanni M . Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution. Proc Natl Acad Sci U S A. 2009; 106(16):6614-9. PMC: 2672516. DOI: 10.1073/pnas.0805957106. View

Remorino A, Korendovych I, Wu Y, DeGrado W, Hochstrasser R . Residue-specific vibrational echoes yield 3D structures of a transmembrane helix dimer. Science. 2011; 332(6034):1206-9. PMC: 3295544. DOI: 10.1126/science.1202997. View

Hahn S, Kim S, Lee C, Cho M . Characteristic two-dimensional IR spectroscopic features of antiparallel and parallel beta-sheet polypeptides: simulation studies. J Chem Phys. 2005; 123(8):084905. DOI: 10.1063/1.1997151. View

10.

Abedini A, Raleigh D . Incorporation of pseudoproline derivatives allows the facile synthesis of human IAPP, a highly amyloidogenic and aggregation-prone polypeptide. Org Lett. 2005; 7(4):693-6. DOI: 10.1021/ol047480+. View

11.

Brewer S, Song B, Raleigh D, Dyer R . Residue specific resolution of protein folding dynamics using isotope-edited infrared temperature jump spectroscopy. Biochemistry. 2007; 46(11):3279-85. DOI: 10.1021/bi602372y. View

12.

Buchanan L, Carr J, Fluitt A, Hoganson A, Moran S, de Pablo J . Structural motif of polyglutamine amyloid fibrils discerned with mixed-isotope infrared spectroscopy. Proc Natl Acad Sci U S A. 2014; 111(16):5796-801. PMC: 4000827. DOI: 10.1073/pnas.1401587111. View

13.

Strasfeld D, Ling Y, Shim S, Zanni M . Tracking fiber formation in human islet amyloid polypeptide with automated 2D-IR spectroscopy. J Am Chem Soc. 2008; 130(21):6698-9. PMC: 3209517. DOI: 10.1021/ja801483n. View

14.

Manor J, Mukherjee P, Lin Y, Leonov H, Skinner J, Zanni M . Gating mechanism of the influenza A M2 channel revealed by 1D and 2D IR spectroscopies. Structure. 2009; 17(2):247-54. PMC: 2820372. DOI: 10.1016/j.str.2008.12.015. View

15.

Moran S, Woys A, Buchanan L, Bixby E, Decatur S, Zanni M . Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils. Proc Natl Acad Sci U S A. 2012; 109(9):3329-34. PMC: 3295317. DOI: 10.1073/pnas.1117704109. View

16.

Marek P, Patsalo V, Green D, Raleigh D . Ionic strength effects on amyloid formation by amylin are a complicated interplay among Debye screening, ion selectivity, and Hofmeister effects. Biochemistry. 2012; 51(43):8478-90. PMC: 3753197. DOI: 10.1021/bi300574r. View

17.

Moran S, Zanni M . How to Get Insight into Amyloid Structure and Formation from Infrared Spectroscopy. J Phys Chem Lett. 2014; 5(11):1984-1993. PMC: 4051309. DOI: 10.1021/jz500794d. View

18.

Williamson J, Miranker A . Direct detection of transient alpha-helical states in islet amyloid polypeptide. Protein Sci. 2006; 16(1):110-7. PMC: 2222845. DOI: 10.1110/ps.062486907. View

19.

Devaraneni P, Komarov A, Costantino C, Devereaux J, Matulef K, Valiyaveetil F . Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state. Proc Natl Acad Sci U S A. 2013; 110(39):15698-703. PMC: 3785774. DOI: 10.1073/pnas.1308699110. View

20.

Huang C, Getahun Z, Wang T, Degrado W, Gai F . Time-resolved infrared study of the helix-coil transition using (13)C-labeled helical peptides. J Am Chem Soc. 2001; 123(48):12111-2. DOI: 10.1021/ja016631q. View