Skip to main content
×
Home
    • Aa
    • Aa

A molecular engineering toolbox for the structural biologist

  • Galia T. Debelouchina (a1) and Tom W. Muir (a1)
Abstract
Abstract

Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

Copyright
Corresponding author
*Author for correspondence: T. W. Muir, Department of Chemistry, Princeton University, Princeton, NJ 08540, USA. Email: muir@princeton.edu
Linked references
Hide All

This list contains references from the content that can be linked to their source. For a full set of references and notes please see the PDF or HTML where available.

N. J. Agard , J. A. Prescher & C. R. Bertozzi (2004). A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. Journal of the American Chemical Society 126(46), 1504615047.

P. Agarwal , R. Kudirka , A. E. Albers , R. M. Barfield , G. W. DE Hart , P. M. Drake , L. C. Jones & D. Rabuka (2013). Hydrazino-Pictet-Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjugate Chemistry 24(6), 846851.

H. W. Ai , W. Shen , A. Sagi , P. R. Chen & P. G. Schultz (2011). Probing protein–protein interactions with a genetically encoded photo-crosslinking amino acid. ChemBioChem 12(12), 18541857.

C. D. Allis & T. W. Muir (2011). Spreading chromatin into chemical biology. ChemBioChem 12(2), 264279.

K. A. Andersen & R. T. Raines (2015). Creating site-specific isopeptide linkages between proteins with the traceless Staudinger ligation. Methods in Molecular Biology 1248, 5565.

J. M. Antos , M. C. Truttmann & H. L. Ploegh (2016). Recent advances in sortase-catalyzed ligation methodology. Current Opinion in Structural Biology 38, 111118.

J. H. Appleby , K. Zhou , G. Volkmann & X. Q. Liu (2009). Novel split intein for trans-splicing synthetic peptide onto C terminus of protein. Journal of Biological Chemistry 284(10), 61946199.

E. Arbely , E. Natan , T. Brandt , M. D. Allen , D. B. Veprintsev , C. V. Robinson , J. W. Chin , A. C. Joerger & A. R. Fersht (2011). Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration. Proceedings of the National Academy of Sciences United States of America 108(20), 82518256.

J. H. Ardenkjaer-Larsen , G. S. Boebinger , A. Comment , S. Duckett , A. S. Edison , F. Engelke , C. Griesinger , R. G. Griffin , C. Hilty , H. Maeda , G. Parigi , T. Prisner , E. Ravera , J. Van Bentum , S. Vega , A. Webb , C. Luchinat , H. Schwalbe & L. Frydman (2015). Facing and overcoming sensitivity challenges in biomolecular NMR spectroscopy. Angewandte Chemie (International edition in English) 54(32), 91629185.

A. Aulabaugh , W. Ding , B. Kapoor , K. Tabei , L. Alksne , R. Dushin , T. Zatz , G. Ellestad & X. Huang (2007). Development of an HPLC assay for Staphylococcus aureus sortase: evidence for the formation of the kinetically competent acyl enzyme intermediate. Analytical Biochemistry 360(1), 1422.

A. Bah & J. D. Forman-Kay (2016). Modulation of intrinsically disordered protein function by post-translational modifications. Journal of Biological Chemistry 291(13), 66966705.

C. R. Baiz , M. Reppert & A. Tokmakoff (2013). An introduction to protein 2D IR spectroscopy. Ultrafast Infrared Vibrational Spectroscopy 361403.

W. A. Barton , D. Tzvetkova-Robev , H. Erdjument-Bromage , P. Tempst & D. B. Nikolov (2006). Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells. Protein Science 15(8), 20082013.

E. Basle , N. Joubert & M. Pucheault (2010). Protein chemical modification on endogenous amino acids. Chemistry & Biology 17(3), 213227.

S. Batjargal , C. R. Walters & E. J. Petersson (2015). Inteins as traceless purification tags for unnatural amino acid proteins. Journal of the American Chemical Society 137(5), 17341737.

M. Beck & W. Baumeister (2016). Cryo-electron tomography: can it reveal the molecular sociology of cells in atomic detail? Trends in Cell Biology 26(11), 825837.

A. Ben-Shem , N. Garreau DE Loubresse , S. Melnikov , L. Jenner , G. Yusupova & M. Yusupov (2011). The structure of the eukaryotic ribosome at 3·0 A resolution. Science 334(6062), 15241529.

G. J. Bernardes , L. Linderoth , K. J. Doores , O. Boutureira & B. G. Davis (2011). Site-selective traceless Staudinger ligation for glycoprotein synthesis reveals scope and limitations. ChemBioChem 12(9), 13831386.

M. Best , A. Degen , M. Baalmann , T. T. Schmidt & R. Wombacher (2015). Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels–Alder reaction. ChemBioChem 16(8), 11581162.

M. L. Blackman , M. Royzen & J. M. Fox (2008). Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. Journal of the American Chemical Society 130(41), 1351813519.

A. F. Brilot , J. Z. Chen , A. Cheng , J. Pan , S. C. Harrison , C. S. Potter , B. Carragher , R. Henderson & N. Grigorieff (2012). Beam-induced motion of vitrified specimen on holey carbon film. Journal of Structural Biology 177(3), 630637.

L. Brocchieri & S. Karlin (2005). Protein length in eukaryotic and prokaryotic proteomes. Nucleic Acids Research 33(10), 33903400.

E. M. Brustad , E. A. Lemke , P. G. Schultz & A. A. Deniz (2008). A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer. Journal of the American Chemical Society 130(52), 1766417665.

D. S. Burz , K. Dutta , D. Cowburn & A. Shekhtman (2006). Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Nature Methods 3(2), 9193.

S. Butterfield , M. Hejjaoui , B. Fauvet , L. Awad & H. A. Lashuel (2012). Chemical strategies for controlling protein folding and elucidating the molecular mechanisms of amyloid formation and toxicity. Journal of Molecular Biology 421(2–3), 204236.

S. D. Cady , K. Schmidt-Rohr , J. Wang , C. S. Soto , W. F. Degrado & M. Hong (2010). Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463(7281), 689692.

M. G. Campbell , D. Veesler , A. Cheng , C. S. Potter & B. Carragher (2015). 2·8 A resolution reconstruction of the thermoplasma acidophilum 20S proteasome using cryo-electron microscopy. Elife 4, e06380.

P. Carvajal-Vallejos , R. Pallisse , H. D. Mootz & S. R. Schmidt (2012). Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. Journal of Biological Chemistry 287(34), 2868628696.

J. Caspi , G. Amitai , O. Belenkiy & S. Pietrokovski (2003). Distribution of split DnaE inteins in cyanobacteria. Molecular Microbiology 50(5), 15691577.

C. Castaneda , J. Liu , A. Chaturvedi , U. Nowicka , T. A. Cropp & D. Fushman (2011a). Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme. Journal of the American Chemical Society 133(44), 1785517868.

C. A. Castaneda , L. Spasser , S. N. Bavikar , A. Brik & D. Fushman (2011b). Segmental isotopic labeling of ubiquitin chains to unravel monomer-specific molecular behavior. Angewandte Chemie (International edition in English) 50(47), 1121011214.

S. E. Cellitti , D. H. Jones , L. Lagpacan , X. Hao , Q. Zhang , H. Hu , S. M. Brittain , A. Brinker , J. Caldwell , B. Bursulaya , G. Spraggon , A. Brock , Y. Ryu , T. Uno , P. G. Schultz & B. H. Geierstanger (2008). In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy. Journal of the American Chemical Society 130(29), 92689281.

K. M. Cergol , R. E. Thompson , L. R. Malins , P. Turner & R. J. Payne (2014). One-pot peptide ligation-desulfurization at glutamate. Organic Letters 16(1), 290293.

A. Chakraborty , A. Mazumder , M. Lin , A. Hasemeyer , Q. Xu , D. Wang , Y. W. Ebright & R. H. Ebright (2015). Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger–Bertozzi ligation. Methods in Molecular Biology 1276, 101131.

J. M. Chalker , G. J. Bernardes , Y. A. Lin & B. G. Davis (2009). Chemical modification of proteins at cysteine: opportunities in chemistry and biology. Chemistry, an Asian Journal 4(5), 630640.

J. M. Chalker , L. Lercher , N. R. Rose , C. J. Schofield & B. G. Davis (2012). Conversion of cysteine into dehydroalanine enables access to synthetic histones bearing diverse post-translational modifications. Angewandte Chemie (International edition in English) 51(8), 18351839.

A. O. Chan , C. M. Ho , H. C. Chong , Y. C. Leung , J. S. Huang , M. K. Wong & C. M. Che (2012). Modification of N-terminal alpha-amino groups of peptides and proteins using ketenes. Journal of the American Chemical Society 134(5), 25892598.

A. Chatterjee , S. B. Sun , J. L. Furman , H. Xiao & P. G. Schultz (2013b). A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli . Biochemistry 52(10), 18281837.

A. Chatterjee , H. Xiao , M. Bollong , H. W. Ai & P. G. Schultz (2013c). Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells. Proceedings of the National Academy of Sciences United States of America 110(29), 1180311808.

C. Chatterjee , R. K. Mcginty , B. Fierz & T. W. Muir (2010). Disulfide-directed histone ubiquitylation reveals plasticity in hDot1L activation. Nature Chemical Biology 6(4), 267269.

H. Chen , S. Viel , F. Ziarelli & L. Peng (2013). 19F NMR: a valuable tool for studying biological events. Chemical Society Reviews 42(20), 79717982.

I. Chen , B. M. Dorr & D. R. Liu (2011). A general strategy for the evolution of bond-forming enzymes using yeast display. Proceedings of the National Academy of Sciences United States of America 108(28), 1139911404.

J. Chen , Y. Ai , J. Wang , L. Haracska & Z. Zhuang (2010a). Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nature Chemical Biology 6(4), 270272.

J. Chen , Q. Wan , Y. Yuan , J. L. Zhu & S. J. Danishefsky (2008). Native chemical ligation at valine: a contribution to peptide and glycopeptide synthesis. Angewandte Chemie (International Edition in English) 47(44), 85218524.

J. Chen , P. Wang , J. L. Zhu , Q. Wan & S. J. Danishefsky (2010b). A program for ligation at threonine sites: application to the controlled total synthesis of glycopeptides. Tetrahedron 66(13), 22772283.

P. R. Chen , D. Groff , J. Guo , W. Ou , S. Cellitti , B. H. Geierstanger & P. G. Schultz (2009). A facile system for encoding unnatural amino acids in mammalian cells. Angewandte Chemie (International edition in English) 48(22), 40524055.

W. N. Chen , K. V. Kuppan , M. D. Lee , K. Jaudzems , T. Huber & G. Otting (2015). O-tert-Butyltyrosine, an NMR tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities. Journal of the American Chemical Society 137(13), 45814586.

Y. Cheng (2015). Single-particle cryo-em at crystallographic resolution. Cell 161(3), 450457.

M. Cheriyan , C. S. Pedamallu , K. Tori & F. Perler (2013). Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. Journal of Biological Chemistry 288(9), 62026211.

J. W. Chin (2014). Expanding and reprogramming the genetic code of cells and animals. Annual Review of Biochemistry 83, 379408.

J. W. Chin , A. B. Martin , D. S. King , L. Wang & P. G. Schultz (2002a). Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli . Proceedings of the National Academy of Sciences United States of America 99(17), 1102011024.

J. W. Chin , S. W. Santoro , A. B. Martin , D. S. King , L. Wang & P. G. Schultz (2002b). Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli . Journal of the American Chemical Society 124(31), 90269027.

C. J. Chou , R. Uprety , L. Davis , J. W. Chin & A. Deiters (2011). Genetically encoding an aliphatic diazirine for protein photocrosslinking. Chemical Science 2(3), 480483.

W. Y. Chow , R. Rajan , K. H. Muller , D. G. Reid , J. N. Skepper , W. C. Wong , R. A. Brooks , M. Green , D. Bihan , R. W. Farndale , D. A. Slatter , C. M. Shanahan & M. J. Duer (2014). NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization. Science 344(6185), 742746.

J. D. Cohen , P. Zou & A. Y. Ting (2012). Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation. ChemBioChem 13(6), 888894.

S. Cohen & E. Arbely (2016). Single-plasmid-based system for efficient noncanonical amino acid mutagenesis in cultured mammalian cells. ChemBioChem 17(11), 10081011.

V. W. Cornish , D. R. Benson , C. A. Altenbach , K. Hideg , W. L. Hubbell & P. G. Schultz (1994). Site-specific incorporation of biophysical probes into proteins. Proceedings of the National Academy of Sciences United States of America 91(8), 29102914.

J. R. Courter , M. Abdo , S. P. Brown , M. J. Tucker , R. M. Hochstrasser & A. B. Smith III (2014). The design and synthesis of alanine-rich alpha-helical peptides constrained by an S,S-tetrazine photochemical trigger: a fragment union approach. Journal of Organic Chemistry 79(2), 759768.

P. Cramer (2016). Structure determination of transient transcription complexes. Biochemical Society Transactions 44(4), 11771182.

D. Crich & A. Banerjee (2007). Native chemical ligation at phenylalanine. Journal of the American Chemical Society 129(33), 10064.

C. N. Cronin , K. B. Lim & J. Rogers (2007). Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells. Protein Science 16(9), 20232029.

G. Cui , S. Park , A. I. Badeaux , D. Kim , J. Lee , J. R. Thompson , F. Yan , S. Kaneko , Z. Yuan , M. V. Botuyan , M. T. Bedford , J. Q. Cheng & G. Mer (2012). PHF20 is an effector protein of p53 double lysine methylation that stabilizes and activates p53. Nature Structural & Molecular Biology 19(9), 916924.

Y. David , M. Vila-Perello , S. Verma & T. W. Muir (2015). Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nature Chemistry 7(5), 394402.

C. M. Davis , A. K. Cooper & R. B. Dyer (2015). Fast helix formation in the B domain of protein A revealed by site-specific infrared probes. Biochemistry 54(9), 17581766.

P. E. Dawson (2011). Native chemical ligation combined with desulfurization and deselenization: a general strategy for chemical protein synthesis. Israel Journal of Chemistry 51(8–9), 862867.

P. E. Dawson & S. B. Kent (2000). Synthesis of native proteins by chemical ligation. Annual Review of Biochemistry 69, 923960.

P. E. Dawson , T. W. Muir , I. Clark-Lewis & S. B. Kent (1994). Synthesis of proteins by native chemical ligation. Science 266(5186), 776779.

G. T. Debelouchina , M. J. Bayro , A. W. Fitzpatrick , V. Ladizhansky , M. T. Colvin , M. A. Caporini , C. P. Jaroniec , V. S. Bajaj , M. Rosay , C. E. Macphee , M. Vendruscolo , W. E. Maas , C. M. Dobson & R. G. Griffin (2013). Higher order amyloid fibril structure by MAS NMR and DNP spectroscopy. Journal of the American Chemical Society 135(51), 1923719247.

G. T. Debelouchina , K. Gerecht & T. W. Muir (2017). Ubiquitin utilizes an acidic surface patch to alter chromatin structure. Nature Chemical Biology 13(1), 105110.

D. J. De Rosier & A. Klug (1968). Reconstruction of three dimensional structures from electron micrographs. Nature 217(5124), 130134.

N. K. Devaraj , R. Weissleder & S. A. Hilderbrand (2008). Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjugate Chemistry 19(12), 22972299.

B. Dhayalan , A. Fitzpatrick , K. Mandal , J. Whittaker , M. A. Weiss , A. Tokmakoff & S. B. Kent (2016). Efficient total chemical synthesis of (13) C=(18) O isotopomers of human insulin for isotope-edited FTIR. ChemBioChem 17(5), 415420.

M. Dimura , T. O. Peulen , C. A. Hanke , A. Prakash , H. Gohlke & C. A. Seidel (2016). Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems. Current Opinion in Structural Biology 40, 163185.

A. Dirksen & P. E. Dawson (2008). Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjugate Chemistry 19(12), 25432548.

J. Dommerholt , S. Schmidt , R. Temming , L. J. Hendriks , F. P. Rutjes , J. C. Van Hest , D. J. Lefeber , P. Friedl & F. L. Van Delft (2010). Readily accessible bicyclononynes for bioorthogonal labeling and three-dimensional imaging of living cells. Angewandte Chemie (International edition in English) 49(49), 94229425.

B. M. Dorr , H. O. Ham , C. An , E. L. Chaikof & D. R. Liu (2014). Reprogramming the specificity of sortase enzymes. Proceedings of the National Academy of Sciences United States of America 111(37), 1334313348.

F. El Oualid , R. Merkx , R. Ekkebus , D. S. Hameed , J. J. Smit , A. De Jong , H. Hilkmann , T. K. Sixma & H. Ovaa (2010). Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angewandte Chemie (International edition in English) 49(52), 1014910153.

S. J. Elsasser , R. J. Ernst , O. S. Walker & J. W. Chin (2016). Genetic code expansion in stable cell lines enables encoded chromatin modification. Nature Methods 13(2), 158164.

A. M. Elsohly & M. B. Francis (2015). Development of oxidative coupling strategies for site-selective protein modification. Accounts of Chemical Research 48(7), 19711978.

P. M. England , Y. Zhang , D. A. Dougherty & H. A. Lester (1999). Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating. Cell 96(1), 8998.

R. J. Ernst , T. P. Krogager , E. S. Maywood , R. Zanchi , V. Beranek , T. S. Elliott , N. P. Barry , M. H. Hastings & J. W. Chin (2016). Genetic code expansion in the mouse brain. Nature Chemical Biology 12(10), 776778.

E. G. Evans & G. L. Millhauser (2015). Genetic incorporation of the unnatural amino acid p-acetyl phenylalanine into proteins for site-directed spin labeling. Methods in Enzymology 563, 503527.

C. Fan , K. Ip & D. Soll (2016). Expanding the genetic code of Escherichia coli with phosphotyrosine. FEBS Letters 590(17), 30403047.

N. L. Fawzi , J. Ying , R. Ghirlando , D. A. Torchia & G. M. Clore (2011). Atomic-resolution dynamics on the surface of amyloid-beta protofibrils probed by solution NMR. Nature 480(7376), 268272.

I. S. Fernandez , X. C. Bai , T. Hussain , A. C. Kelley , J. R. Lorsch , V. Ramakrishnan & S. H. Scheres (2013). Molecular architecture of a eukaryotic translational initiation complex. Science 342(6160), 1240585.

S. Ficht , R. J. Payne , A. Brik & C. H. Wong (2007). Second-generation sugar-assisted ligation: a method for the synthesis of cysteine-containing glycopeptides. Angewandte Chemie (International edition in English) 46(31), 59755979.

A. W. Fitzpatrick , G. T. Debelouchina , M. J. Bayro , D. K. Clare , M. A. Caporini , V. S. Bajaj , C. P. Jaroniec , L. Wang , V. Ladizhansky , S. A. Muller , C. E. Macphee , C. A. Waudby , H. R. Mott , A. De Simone , T. P. Knowles , H. R. Saibil , M. Vendruscolo , E. V. Orlova , R. G. Griffin & C. M. Dobson (2013). Atomic structure and hierarchical assembly of a cross-beta amyloid fibril. Proceedings of the National Academy of Sciences United States of America 110(14), 54685473.

K. K. Frederick , V. K. Michaelis , B. Corzilius , T. C. Ong , A. C. Jacavone , R. G. Griffin & S. Lindquist (2015). Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus. Cell 163(3), 620628.

D. I. Freedberg & P. Selenko (2014). Live cell NMR. Annual Review of Biophysics 43, 171192.

L. Freiburger , M. Sonntag , J. Hennig , J. Li , P. Zou & M. Sattler (2015). Efficient segmental isotope labeling of multi-domain proteins using Sortase A. Journal of Biomolecular NMR 63(1), 18.

S. Frutos , M. Goger , B. Giovani , D. Cowburn & T. W. Muir (2010). Branched intermediate formation stimulates peptide bond cleavage in protein splicing. Nature Chemical Biology 6(7), 527533.

D. P. Gamblin , S. Van Kasteren , G. J. Bernardes , J. M. Chalker , N. J. Oldham , A. J. Fairbanks & B. G. Davis (2008). Chemical site-selective prenylation of proteins. Molecular BioSystems 4(6), 558561.

M. J. Gattner , M. Vrabel & T. Carell (2013). Synthesis of epsilon-N-propionyl-, epsilon-N-butyryl-, and epsilon-N-crotonyl-lysine containing histone H3 using the pyrrolysine system. Chemical Communications (Cambridge) 49(4), 379381.

S. George , J. D. Aguirre , D. E. Spratt , Y. Bi , M. Jeffery , G. S. Shaw & P. O'donoghue (2016). Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping. FEBS Letters 590(10), 15301542.

M. A. Gilles , A. Q. Hudson & C. L. Borders Jr. (1990). Stability of water-soluble carbodiimides in aqueous solution. Analytical Biochemistry 184(2), 244248.

J. E. Glasgow , M. L. Salit & J. R. Cochran (2016). In vivo site-specific protein tagging with diverse amines using an engineered sortase variant. Journal of the American Chemical Society 138(24), 74967499.

Y. Goto , T. Katoh & H. SUGA (2011). Flexizymes for genetic code reprogramming. Nature Protocols 6(6), 779790.

E. J. Grayson , S. J. Ward , A. L. Hall , P. M. Rendle , D. P. Gamblin , A. S. Batsanov & B. G. Davis (2005). Glycosyl disulfides: novel glycosylating reagents with flexible aglycon alteration. Journal of Organic Chemistry 70(24), 97409754.

S. Greiss & J. W. Chin (2011). Expanding the genetic code of an animal. Journal of the American Chemical Society 133(36), 1419614199.

B. A. Griffin , S. R. Adams & R. Y. Tsien (1998). Specific covalent labeling of recombinant protein molecules inside live cells. Science 281(5374), 269272.

W. Grosse , L. O. Essen & U. Koert (2011). Strategies and perspectives in ion-channel engineering. ChemBioChem 12(6), 830839.

D. Guan , M. Ramirez & Z. Chen (2013). Split intein mediated ultra-rapid purification of tagless protein (SIRP). Biotechnology and Bioengineering 110(9), 24712481.

C. P. Guimaraes , M. D. Witte , C. S. Theile , G. Bozkurt , L. Kundrat , A. E. Blom & H. L. Ploegh (2013). Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nature Protocols 8(9), 17871799.

C. Haase , H. Rohde & O. Seitz (2008). Native chemical ligation at valine. Angewandte Chemie (International edition in English) 47(36), 68076810.

S. M. Hancock , R. Uprety , A. Deiters & J. W. Chin (2010). Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. Journal of the American Chemical Society 132(42), 1481914824.

C. M. Haney , R. F. Wissner & E. J. Petersson (2015). Multiply labeling proteins for studies of folding and stability. Current Opinion in Chemical Biology 28, 123130.

C. M. Haney , R. F. Wissner , J. B. Warner , Y. J. Wang , J. J. Ferrie , D. J. Covell , R. J. Karpowicz , V. M. LEE & E. J. Petersson (2016). Comparison of strategies for non-perturbing labeling of alpha-synuclein to study amyloidogenesis. Organic & Biomolecular 14(5), 15841592.

T. J. Harmand , C. E. Murar & J. W. Bode (2014). New chemistries for chemoselective peptide ligations and the total synthesis of proteins. Current Opinion in Chemical Biology 22, 115121.

Z. Harpaz , P. Siman , K. S. A. Kumar & A. Brik (2010). Protein synthesis assisted by native chemical ligation at leucine. ChemBioChem 11(9), 12321235.

S. Hauke , M. Best , T. T. Schmidt , M. Baalmann , A. Krause & R. Wombacher (2014). Two-step protein labeling utilizing lipoic acid ligase and Sonogashira cross-coupling. Bioconjugate Chemistry 25(9), 16321637.

Y. He , J. Fang , D. J. Taatjes & E. Nogales (2013). Structural visualization of key steps in human transcription initiation. Nature 495(7442), 481486.

M. Hejjaoui , S. Butterfield , B. Fauvet , F. Vercruysse , J. Cui , I. Dikiy , M. Prudent , D. Olschewski , Y. ZHANG , D. Eliezer & H. A. Lashuel (2012). Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: alpha-synuclein phosphorylation at tyrosine 125. Journal of the American Chemical Society 134(11), 51965210.

H. Hirakawa , S. Ishikawa & T. Nagamune (2015). Ca2+ -independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli . Biotechnol Journal 10(9), 14871492.

C. Hoffmann , G. Gaietta , M. Bunemann , S. R. Adams , S. Oberdorff-Maass , B. Behr , J. P. Vilardaga , R. Y. Tsien , M. H. Ellisman & M. J. Lohse (2005). A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nature Methods 2(3), 171176.

A. N. Holding (2015). XL-MS: protein cross-linking coupled with mass spectrometry. Methods 89, 5463.

Q. Y. Hu , F. Berti & R. Adamo (2016). Towards the next generation of biomedicines by site-selective conjugation. Chemical Society Reviews 45(6), 16911719.

T. Huber , S. Naganathan , H. Tian , S. Ye & T. P. Sakmar (2013). Unnatural amino acid mutagenesis of GPCRs using amber codon suppression and bioorthogonal labeling. Methods in Enzymology 520, 281305.

N. Huguenin-Dezot , V. De Cesare , J. Peltier , A. Knebel , Y. A. Kristaryianto , D. T. Rogerson , Y. Kulathu , M. Trost & J. W. Chin (2016). Synthesis of isomeric phosphoubiquitin chains reveals that phosphorylation controls deubiquitinase activity and specificity. Cell Reports 16(4), 11801193.

F. Husada , G. Gouridis , R. Vietrov , G. K. Schuurman-Wolters , E. Ploetz , M. De Boer , B. Poolman & T. Cordes (2015). Watching conformational dynamics of ABC transporters with single-molecule tools. Biochemical Society Transactions 43(5), 10411047.

A. A. Hyman , C. A. Weber & F. Julicher (2014). Liquid-liquid phase separation in biology. Annual Review of Cell and Developmental Biology 30, 3958.

T. I. Igumenova , A. E. Mcdermott , K. W. Zilm , R. W. Martin , E. K. Paulson & A. J. Wand (2004). Assignments of carbon NMR resonances for microcrystalline ubiquitin. Journal of the American Chemical Society 126(21), 67206727.

K. Inomata , A. Ohno , H. Tochio , S. Isogai , T. Tenno , I. Nakase , T. Takeuchi , S. Futaki , Y. Ito , H. Hiroaki & M. Shirakawa (2009). High-resolution multi-dimensional NMR spectroscopy of proteins in human cells. Nature 458(7234), 106109.

H. Iwai , S. zuger , J. Jin & P. H. Tam (2006). Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme . FEBS Letters 580(7), 18531858.

J. C. Jackson , J. T. Hammill & R. A. Mehl (2007). Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity. Journal of the American Chemical Society 129(5), 11601166.

D. B. Johnson , J. Xu , Z. Shen , J. K. Takimoto , M. D. Schultz , R. J. Schmitz , Z. Xiang , J. R. Ecker , S. P. Briggs & L. Wang (2011). RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites. Nature Chemical Biology 7(11), 779786.

D. H. Jones , S. E. Cellitti , X. Hao , Q. Zhang , M. Jahnz , D. Summerer , P. G. Schultz , T. Uno & B. H. Geierstanger (2010). Site-specific labeling of proteins with NMR-active unnatural amino acids. Journal of Biomolecular NMR 46(1), 89100.

N. S. Joshi , L. R. Whitaker & M. B. Francis (2004). A three-component Mannich-type reaction for selective tyrosine bioconjugation. Journal of the American Chemical Society 126(49), 1594215943.

J. K. Judice , T. R. Gamble , E. C. Murphy , A. M. De Vos & P. G. Schultz (1993). Probing the mechanism of staphylococcal nuclease with unnatural amino acids: kinetic and structural studies. Science 261(5128), 15781581.

S. Kalkhof & A. Sinz (2008). Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters. Analytical and Bioanalytical Chemistry 392(1–2), 305312.

P. M. Kane , C. T. Yamashiro , D. F. Wolczyk , N. Neff , M. Goebl & T. H. Stevens (1990). Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science 250(4981), 651657.

J. C. Kendrew , G. Bodo , H. M. Dintzis , R. G. Parrish , H. Wyckoff & D. C. Phillips (1958). A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature 181(4610), 662666.

S. Kent , Y. Sohma , S. Liu , D. Bang , B. Pentelute & K. Mandal (2012). Through the looking glass--a new world of proteins enabled by chemical synthesis. Journal of Peptide Science 18(7), 428436.

C. H. Kim , M. Kang , H. J. Kim , A. Chatterjee & P. G. Schultz (2012). Site-specific incorporation of epsilon-N-crotonyllysine into histones. Angewandte Chemie (International edition in English) 51(29), 72467249.

Y. Kobashigawa , H. Kumeta , K. Ogura & F. Inagaki (2009). Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method. Journal of Biomolecular NMR 43(3), 145150.

C. Koehler , P. F. Sauter , M. Wawryszyn , G. E. Girona , K. Gupta , J. J. Landry , M. H. Fritz , K. Radic , J. E. Hoffmann , Z. A. Chen , J. Zou , P. S. Tan , B. Galik , S. Junttila , P. Stolt-Bergner , G. Pruneri , A. GYENESEI , C. Schultz , M. B. Biskup , H. Besir , V. Benes , J. Rappsilber , M. Jechlinger , J. O. Korbel , I. Berger , S. Braese & E. A. Lemke (2016). Genetic code expansion for multiprotein complex engineering. Nature Methods 13, 9971000.

J. T. Koh , V. W. Cornish & P. G. Schultz (1997). An experimental approach to evaluating the role of backbone interactions in proteins using unnatural amino acid mutagenesis. Biochemistry 36(38), 1131411322.

C. Kohrer , J. H. Yoo , M. Bennett , J. Schaack & U. L. Rajbhandary (2003). A possible approach to site-specific insertion of two different unnatural amino acids into proteins in mammalian cells via nonsense suppression. Chemistry & Biology 10(11), 10951102.

H. C. Kolb , M. G. Finn & K. B. Sharpless (2001). Click chemistry: diverse chemical function from a few good reactions. Angewandte Chemie (International edition in English) 40(11), 20042021.

S. Kucher , S. Korneev , S. Tyagi , R. Apfelbaum , D. Grohmann , E. A. Lemke , J. P. Klare , H. J. Steinhoff & D. Klose (2016). Orthogonal spin labeling using click chemistry for in vitro and in vivo applications. Journal of Magnetic Resonance 275, 3845.

N. Kuhlmann , S. Wroblowski , P. Knyphausen , S. DE Boor , J. Brenig , A. Y. Zienert , K. Meyer-Teschendorf , G. J. Praefcke , H. Nolte , M. Kruger , M. SCHACHERL , U. Baumann , L. C. James , J. W. Chin & M. Lammers (2016). Structural and mechanistic insights into the regulation of the fundamental rho regulator rhogdialpha by lysine acetylation. Journal of Biological Chemistry 291(11), 54845499.

S. M. Kuhn , M. Rubini , M. A. Muller & A. Skerra (2011). Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore. Journal of the American Chemical Society 133(11), 37083711.

K. S. A. Kumar , M. Haj-Yahya , D. Olschewski , H. A. Lashuel & A. Brik (2009). Highly efficient and chemoselective peptide ubiquitylation. Angewandte Chemie (International edition in English) 48(43), 80908094.

B. Kwon , D. Tietze , P. B. White , S. Y. Liao & M. Hong (2015). Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain. Protein Science 24(7), 10871099.

I. Kwon , P. Wang & D. A. Tirrell (2006). Design of a bacterial host for site-specific incorporation of p-bromophenylalanine into recombinant proteins. Journal of the American Chemical Society 128(36), 1177811783.

M. J. Lajoie , A. J. Rovner , D. B. Goodman , H. R. Aerni , A. D. Haimovich , G. Kuznetsov , J. A. Mercer , H. H. Wang , P. A. Carr , J. A. Mosberg , N. Rohland , P. G. Schultz , J. M. Jacobson , J. Rinehart , G. M. Church & F. J. Isaacs (2013). Genomically recoded organisms expand biological functions. Science 342(6156), 357360.

M. Lammers , H. Neumann , J. W. Chin & L. C. James (2010). Acetylation regulates cyclophilin A catalysis, immunosuppression and HIV isomerization. Nature Chemical Biology 6(5), 331337.

K. Lang , L. Davis , J. torres-Kolbus , C. Chou , A. Deiters & J. W. Chin (2012a). Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nature Chemistry 4(4), 298304.

K. Lang , L. Davis , S. Wallace , M. Mahesh , D. J. Cox , M. L. Blackman , J. M. Fox & J. W. Chin (2012b). Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions. Journal of the American Chemical Society 134(25), 1031710320.

S. T. Laughlin , J. M. Baskin , S. L. Amacher & C. R. Bertozzi (2008). In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320(5876), 664667.

T. Lavergne , R. Lamichhane , D. A. Malyshev , Z. Li , L. Li , E. Sperling , J. R. Williamson , D. P. Millar & F. E. Romesberg (2016). FRET characterization of complex conformational changes in a large 16S ribosomal RNA fragment site-specifically labeled using unnatural base pairs. ACS Chemical Biology 11(5), 13471353.

H. S. Lee , J. Guo , E. A. Lemke , R. D. Dimla & P. G. Schultz (2009a). Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae . Journal of the American Chemical Society 131(36), 1292112923.

H. S. Lee , G. Spraggon , P. G. Schultz & F. Wang (2009b). Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe. Journal of the American Chemical Society 131(7), 24812483.

G. A. Lemieux , C. L. De Graffenried & C. R. Bertozzi (2003). A fluorogenic dye activated by the staudinger ligation. Journal of the American Chemical Society 125(16), 47084709.

K. R. Lennard & A. Tavassoli (2014). Peptides come round: using SICLOPPS libraries for early stage drug discovery. Chemistry 20(34), 1060810614.

A. L. Le Sueur , R. E. Horness & M. C. Thielges (2015). Applications of two-dimensional infrared spectroscopy. Analyst 140(13), 43364349.

D. Li , V. E. Pye & M. Caffrey (2015). Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method. Acta Crystallographica D, Biological Crystallography 71(Pt 1), 104122.

F. Li , P. Shi , J. Li , F. Yang , T. Wang , W. Zhang , F. Gao , W. Ding , D. Li , J. Li , Y. Xiong , J. Sun , W. Gong , C. Tian & J. Wang (2013a). A genetically encoded 19F NMR probe for tyrosine phosphorylation. Angewandte Chemie (International edition in English) 52(14), 39583962.

F. Li , H. Zhang , Y. Sun , Y. Pan , J. Zhou & J. Wang (2013b). Expanding the genetic code for photoclick chemistry in E. coli, mammalian cells, and A. thaliana . Angewandte Chemie (International edition in English) 52(37), 97009704.

S. Lin , X. Yang , S. Jia , A. M. Weeks , M. Hornsby , P. S. LEE , R. V. Nichiporuk , A. T. Iavarone , J. A. Wells , F. D. Toste & C. J. Chang (2017). Redox-based reagents for chemoselective methionine bioconjugation. Science 355(6325), 597602.

A. J. Link , M. K. Vink & D. A. Tirrell (2004). Presentation and detection of azide functionality in bacterial cell surface proteins. Journal of the American Chemical Society 126(34), 1059810602.

C. C. Liu , E. Brustad , W. LIU & P. G. Schultz (2007). Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin. Journal of the American Chemical Society 129(35), 1064810649.

C. C. Liu & P. G. Schultz (2010). Adding new chemistries to the genetic code. Annual Review of Biochemistry 79, 413444.

D. S. Liu , A. Tangpeerachaikul , R. Selvaraj , M. T. Taylor , J. M. Fox & A. Y. Ting (2012). Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells. Journal of the American Chemical Society 134(2), 792795.

F. Liu , E. Y. Luo , D. B. Flora & A. R. Mezo (2014a). Irreversible sortase A-mediated ligation driven by diketopiperazine formation. Journal of Organic Chemistry 79(2), 487492.

Z. Liu , S. Frutos , M. J. Bick , M. Vila-Perello , G. T. Debelouchina , S. A. Darst & T. W. Muir (2014b). Structure of the branched intermediate in protein splicing. Proceedings of the National Academy of Sciences United States of America 111(23), 84228427.

K. V. Loscha , A. J. Herlt , R. Qi , T. Huber , K. Ozawa & G. Otting (2012). Multiple-site labeling of proteins with unnatural amino acids. Angewandte Chemie (International edition in English) 51(9), 22432246.

P. Lossl , M. Van De Waterbeemd & A. J. Heck (2016). The diverse and expanding role of mass spectrometry in structural and molecular biology. EMBO Journal 35(24), 26342657.

J. X. Lu , W. Qiang , W. M. Yau , C. D. Schwieters , S. C. Meredith & R. Tycko (2013). Molecular structure of beta-amyloid fibrils in Alzheimer's disease brain tissue. Cell 154(6), 12571268.

W. Lu , M. Randal , A. Kossiakoff & S. B. Kent (1999). Probing intermolecular backbone H-bonding in serine proteinase-protein inhibitor complexes. Chemistry & Biology 6(7), 419427.

X. Lu , M. D. Simon , J. V. Chodaparambil , J. C. Hansen , K. M. Shokat & K. Luger (2008). The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structure. Nature Structural & Molecular Biology 15(10), 11221124.

C. Ludwig , M. Pfeiff , U. Linne & H. D. Mootz (2006). Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing. Angewandte Chemie (International edition in English) 45(31), 52185221.

R. P. Lyon , J. R. Setter , T. D. Bovee , S. O. Doronina , J. H. Hunter , M. E. Anderson , C. L. Balasubramanian , S. M. Duniho , C. I. Leiske , F. Li & P. D. Senter (2014). Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nature Biotechnology 32(10), 10591062.

J. I. Macdonald , H. K. Munch , T. Moore & M. B. Francis (2015). One-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes. Nature Chemical Biology 11(5), 326331.

D. Macmillan , R. M. Bill , K. A. Sage , D. Fern & S. L. Flitsch (2001). Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin. Chemistry & Biology 8(2), 133145.

L. R. Malins , K. M. Cergol & R. J. Payne (2013). Peptide ligation-desulfurization chemistry at Arginine. ChemBioChem 14(5), 559563.

L. R. Malins , K. M. Cergol & R. J. Payne (2014). Chemoselective sulfenylation and peptide ligation at tryptophan. Chemical Science 5(1), 260266.

L. R. Malins & R. J. Payne (2015). Modern extensions of native chemical ligation for chemical protein synthesis. Topics in Current Chemistry 362, 2787.

A. Mallagaray , G. Dominguez , T. Peters & J. Perez-Castells (2016). A rigid lanthanide binding tag to aid NMR studies of a 70 kDa homodimeric coat protein of human norovirus. Chemical Communications (Cambridge) 52(3), 601604.

T. Maly , G. T. Debelouchina , V. S. Bajaj , K. N. Hu , C. G. Joo , M. L. Mak-Jurkauskas , J. R. Sirigiri , P. C. Van Der Wel , J. Herzfeld , R. J. Temkin & R. G. Griffin (2008). Dynamic nuclear polarization at high magnetic fields. Journal of Chemical Physics 128(5), 052211.

D. A. Malyshev , K. Dhami , T. Lavergne , T. Chen , N. Dai , J. M. Foster , I. R. Correa Jr. & F. E. Romesberg (2014). A semi-synthetic organism with an expanded genetic alphabet. Nature 509(7500), 385388.

K. Mandal , M. Uppalapati , D. Ault-Riche , J. Kenney , J. Lowitz , S. S. Sidhu & S. B. Kent (2012). Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography. Proceedings of the National Academy of Sciences United States of America 109(37), 1477914784.

A. Mandlik , A. Swierczynski , A. Das & H. Ton-That (2008). Pili in Gram-positive bacteria: assembly, involvement in colonization and biofilm development. Trends in Microbiology 16(1), 3340.

H. Mao , S. A. Hart , A. Schink & B. A. Pollok (2004). Sortase-mediated protein ligation: a new method for protein engineering. Journal of the American Chemical Society 126(9), 26702671.

J. Marecek , B. Song , S. Brewer , J. Belyea , R. B. Dyer & D. P. Raleigh (2007). A simple and economical method for the production of 13C,18O-labeled Fmoc-amino acids with high levels of enrichment: applications to isotope-edited IR studies of proteins. Organic Letters 9(24), 49354937.

E. N. Marsh & Y. Suzuki (2014). Using (19)F NMR to probe biological interactions of proteins and peptides. ACS Chemical Biology 9(6), 12421250.

C. Martinez , P. De Geus , P. Stanssens , M. Lauwereys & C. Cambillau (1993). Engineering cysteine mutants to obtain crystallographic phases with a cutinase from Fusarium solani pisi . Protein Engineering 6(2), 157165.

D. Matthies , O. Dalmas , M. J. Borgnia , P. K. Dominik , A. Merk , P. Rao , B. G. Reddy , S. Islam , A. Bartesaghi , E. Perozo & S. Subramaniam (2016). Cryo-EM structures of the magnesium channel CorA reveal symmetry break upon gating. Cell 164(4), 747756.

S. K. Mazmanian , G. Liu , H. Ton-That & O. Schneewind (1999). Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285(5428), 760763.

J. M. Mcfarland & M. B. Francis (2005). Reductive alkylation of proteins using iridium catalyzed transfer hydrogenation. Journal of the American Chemical Society 127(39), 1349013491.

J. M. Mcfarland , N. S. Joshi & M. B. Francis (2008). Characterization of a three-component coupling reaction on proteins by isotopic labeling and nuclear magnetic resonance spectroscopy. Journal of the American Chemical Society 130(24), 76397644.

R. K. Mcginty & S. Tan (2015). Nucleosome structure and function. Chemical Reviews 115(6), 22552273.

F. Meier , T. Abeywardana , A. Dhall , N. P. Marotta , J. Varkey , R. Langen , C. Chatterjee & M. R. Pratt (2012). Semisynthetic, site-specific ubiquitin modification of alpha-synuclein reveals differential effects on aggregation. Journal of the American Chemical Society 134(12), 54685471.

A. Merk , A. Bartesaghi , S. Banerjee , V. Falconieri , P. Rao , M. I. Davis , R. Pragani , M. B. Boxer , L. A. Earl , J. L. Milne & S. Subramaniam (2016). Breaking Cryo-EM resolution barriers to facilitate drug discovery. Cell 165(7), 16981707.

E. Mileo , E. Etienne , M. Martinho , R. Lebrun , V. Roubaud , P. Tordo , B. Gontero , B. Guigliarelli , S. R. Marque & V. Belle (2013). Enlarging the panoply of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR): sensitive and selective spin-labeling of tyrosine using an isoindoline-based nitroxide. Bioconjugate Chemistry 24(6), 11101117.

S. Milles , S. Tyagi , N. Banterle , C. Koehler , V. Vandelinder , T. Plass , A. P. Neal & E. A. Lemke (2012). Click strategies for single-molecule protein fluorescence. Journal of the American Chemical Society 134(11), 51875195.

M. Minoshima & K. Kikuchi (2017). Photostable and photoswitching fluorescent dyes for super-resolution imaging. Journal of Biological Inorganic Chemistry. doi: 10.1007/s00775-016-1435-y.

H. D. Mootz , E. S. Blum , A. B. Tyszkiewicz & T. W. Muir (2003). Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo . Journal of the American Chemical Society 125(35), 1056110569.

S. D. Moran , A. M. Woys , L. E. Buchanan , E. Bixby , S. M. Decatur & M. T. Zanni (2012). Two-dimensional IR spectroscopy and segmental C-13 labeling reveals the domain structure of human gamma D-crystallin amyloid fibrils. Proceedings of the National Academy of Sciences of the United States of America 109(9), 33293334.

M. T. Morgan , M. Haj-Yahya , A. E. Ringel , P. Bandi , A. Brik & C. Wolberger (2016). Structural basis for histone H2B deubiquitination by the SAGA DUB module. Science 351(6274), 725728.

T. Moyal , H. P. Hemantha , P. Siman , M. Refua & A. Brik (2013). Highly efficient one-pot ligation and desulfurization. Chemical Science 4(6), 24962501.

T. W. Muir , D. Sondhi & P. A. Cole (1998). Expressed protein ligation: a general method for protein engineering. Proceedings of the National Academy of Sciences United States of America 95(12), 67056710.

T. Mukai , A. Hayashi , F. Iraha , A. Sato , K. Ohtake , S. Yokoyama & K. Sakamoto (2010a). Codon reassignment in the Escherichia coli genetic code. Nucleic Acids Research 38(22), 81888195.

F. Munari , S. Soeroes , H. M. Zenn , A. Schomburg , N. Kost , S. Schroder , R. Klingberg , N. Rezaei-Ghaleh , A. Stutzer , K. A. Gelato , P. J. Walla , S. Becker , D. Schwarzer , B. Zimmermann , W. Fischle & M. Zweckstetter (2012). Methylation of lysine 9 in histone H3 directs alternative modes of highly dynamic interaction of heterochromatin protein hHP1beta with the nucleosome. Journal of Biological Chemistry 287(40), 3375633765.

M. Muona , A. S. Aranko , V. Raulinaitis & H. Iwai (2010). Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro . Nature Protocols 5(3), 574587.

V. Muralidharan & T. W. Muir (2006). Protein ligation: an enabling technology for the biophysical analysis of proteins. Nature Methods 3(6), 429438.

M. T. Naik , N. Suree , U. Ilangovan , C. K. Liew , W. Thieu , D. O. Campbell , J. J. Clemens , M. E. Jung & R. T. Clubb (2006). Staphylococcus aureus Sortase A transpeptidase. Calcium promotes sorting signal binding by altering the mobility and structure of an active site loop. Journal of Biological Chemistry 281(3), 18171826.

T. Nakamura , Y. Kawai , N. Kitamoto , T. Osawa & Y. Kato (2009). Covalent modification of lysine residues by allyl isothiocyanate in physiological conditions: plausible transformation of isothiocyanate from thiol to amine. Chem Res Toxicol 22(3), 536542.

J. E. Nettleship , R. Assenberg , J. M. Diprose , N. Rahman-Huq & R. J. Owens (2010). Recent advances in the production of proteins in insect and mammalian cells for structural biology. Journal of Structural Biology 172(1), 5565.

H. Neumann , S. Y. Peak-Chew & J. W. Chin (2008). Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nature Chemical Biology 4(4), 232234.

H. Neumann , K. Wang , L. Davis , M. Garcia-Alai & J. W. Chin (2010). Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464(7287), 441444.

P. Neumann-Staubitz & H. Neumann (2016). The use of unnatural amino acids to study and engineer protein function. Current Opinion in Structural Biology 38, 119128.

R. Neutze , G. Branden & G. F. Schertler (2015). Membrane protein structural biology using X-ray free electron lasers. Current Opinion in Structural Biology 33, 115125.

D. P. Nguyen , T. Elliott , M. Holt , T. W. Muir & J. W. Chin (2011). Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. Journal of the American Chemical Society 133(30), 1141811421.

D. P. Nguyen , M. M. Garcia Alai , S. Virdee & J. W. Chin (2010). Genetically directing varepsilon-N, N-dimethyl-L-lysine in recombinant histones. Chemistry & Biology 17(10), 10721076.

G. K. Nguyen , A. Kam , S. Loo , A. E. Jansson , L. X. Pan & J. P. Tam (2015). Butelase 1: a versatile ligase for peptide and protein macrocyclization. Journal of the American Chemical Society 137(49), 1539815401.

G. K. Nguyen , S. Wang , Y. Qiu , X. Hemu , Y. Lian & J. P. Tam (2014). Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nature Chemical Biology 10(9), 732738.

I. Nikic & E. A. Lemke (2015). Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Current Opinion in Chemical Biology 28, 164173.

B. L. Nilsson , L. L. Kiessling & R. T. Raines (2000). Staudinger ligation: a peptide from a thioester and azide. Organic Letters 2(13), 19391941.

E. Nogales (2016). The development of cryo-EM into a mainstream structural biology technique. Nature Methods 13(1), 2427.

C. J. Noren , S. J. Anthony-cahill , M. C. Griffith & P. G. Schultz (1989). A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244(4901), 182188.

N. Ostrov , M. Landon , M. Guell , G. Kuznetsov , J. Teramoto , N. Cervantes , M. Zhou , K. Singh , M. G. Napolitano , M. Moosburner , E. Shrock , B. W. Pruitt , N. Conway , D. B. Goodman , C. L. Gardner , G. Tyree , A. Gonzales , B. L. Wanner , J. E. Norville , M. J. Lajoie & G. M. Church (2016). Design, synthesis, and testing toward a 57-codon genome. Science 353(6301), 819822.

M. Pan , S. Gao , Y. Zheng , X. Tan , H. Lan , X. Tan , D. Sun , L. Lu , T. Wang , Q. Zheng , Y. Huang , J. Wang & L. LIU (2016). Quasi-racemic X-ray structures of K27-linked ubiquitin chains prepared by total chemical synthesis. Journal of the American Chemical Society 138(23), 74297435.

S. H. Park , V. S. Wang , J. Radoicic , A. A. De Angelis , S. Berkamp & S. J. Opella (2015). Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA). Journal of Biomolecular NMR, 61(3–4), 185196.

B. L. Pentelute & S. B. H. Kent (2007). Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation. Organic Letters 9(4), 687690.

L. Perdios , A. R. Lowe , G. Saladino , T. D. Bunney , N. Thiyagarajan , Y. Alexandrov , C. Dunsby , P. M. French , J. W. Chin , F. L. Gervasio , E. W. Tate & M. Katan (2017). Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET. Science Report 7, 39841.

F. B. Perler (2002). InBase: the intein database. Nucleic Acids Research 30(1), 383384.

K. Pervushin , R. Riek , G. Wider & K. Wuthrich (1997). Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proceedings of the National Academy of Sciences United States of America 94(23), 1236612371.

A. T. Petkova , W. M. Yau & R. Tycko (2006). Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils. Biochemistry 45(2), 498512.

S. Pietrokovski (2001). Intein spread and extinction in evolution. Trends in Genetics 17(8), 465472.

A. C. Pike , E. F. Garman , T. Krojer , F. Von Delft & E. P. Carpenter (2016). An overview of heavy-atom derivatization of protein crystals. Acta Crystallographica D, Biological Crystallography 72(Pt 3), 303318.

J. G. Plaks , R. Falatach , M. Kastantin , J. A. Berberich & J. L. Kaar (2015). Multisite clickable modification of proteins using lipoic acid ligase. Bioconjugate Chemistry 26(6), 11041112.

C. Plaschka , M. Hantsche , C. Dienemann , C. Burzinski , J. Plitzko & P. Cramer (2016). Transcription initiation complex structures elucidate DNA opening. Nature 533(7603), 353358.

T. Plass , S. Milles , C. Koehler , C. Schultz & E. A. Lemke (2011). Genetically encoded copper-free click chemistry. Angewandte Chemie (International edition in English) 50(17), 38783881.

R. L. Policarpo , H. Kang , X. Liao , A. E. Rabideau , M. D. Simon & B. L. Pentelute (2014). Flow-based enzymatic ligation by sortase A. Angewandte Chemie (International edition in English) 53(35), 92039208.

S. J. Pollack & P. G. Schultz (1989). A semisynthetic catalytic antibody. Journal of the American Chemical Society 111(5), 19291931.

M. W. Popp & H. L. Ploegh (2011). Making and breaking peptide bonds: protein engineering using sortase. Angewandte Chemie (International edition in English) 50(22), 50245032.

F. Rabanal , W. F. Degrado & P. L. Dutton (1996). Use of 2,2′-dithiobis(5-nitropyridine) for the heterodimerization of cysteine containing peptides. Introduction of the 5-nitro-2-pyridinesulfenyl group. Tetrahedron Letters 37(9), 13471350.

C. Ratzke , B. Hellenkamp & T. Hugel (2014). Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery. Nature Communications 5, 4192.

P. S. Reddy , S. Dery & N. Metanis (2016). Chemical synthesis of proteins with non-strategically placed cysteines using selenazolidine and selective deselenization. Angewandte Chemie (International edition in English) 55(3), 992995.

M. Ritzefeld (2014). Sortagging: a robust and efficient chemoenzymatic ligation strategy. Chemistry 20(28), 85168529.

D. T. Rogerson , A. Sachdeva , K. Wang , T. Haq , A. Kazlauskaite , S. M. Hancock , N. Huguenin-Dezot , M. M. Muqit , A. M. Fry , R. Bayliss & J. W. Chin (2015). Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog. Nature Chemical Biology 11(7), 496503.

V. V. Rostovtsev , L. G. Green , V. V. Fokin & K. B. Sharpless (2002). A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective ‘ligation’ of azides and terminal alkynes. Angewandte Chemie (International edition in English) 41(14), 25962599.

A. Sachdeva , K. Wang , T. Elliott & J. W. Chin (2014). Concerted, rapid, quantitative, and site-specific dual labeling of proteins. Journal of the American Chemical Society 136(22), 77857788.

D. Sakakibara , A. Sasaki , T. Ikeya , J. Hamatsu , T. Hanashima , M. Mishima , M. Yoshimasu , N. Hayashi , T. Mikawa , M. Walchli , B. O. Smith , M. Shirakawa , P. Guntert & Y. Ito (2009). Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458(7234), 102105.

K. Sakamoto , K. Murayama , K. Oki , F. Iraha , M. Kato-Murayama , M. Takahashi , K. Ohtake , T. Kobayashi , S. Kuramitsu , M. Shirouzu & S. Yokoyama (2009). Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography. Structure 17(3), 335344.

E. Saxon , J. I. Armstrong & C. R. Bertozzi (2000). A ‘traceless’ Staudinger ligation for the chemoselective synthesis of amide bonds. Organic Letters 2(14), 21412143.

E. Saxon & C. R. Bertozzi (2000). Cell surface engineering by a modified Staudinger reaction. Science 287(5460), 20072010.

J. Sayers , R. E. Thompson , K. J. Perry , L. R. Malins & R. J. Payne (2015). Thiazolidine-protected beta-thiol asparagine: applications in one-pot ligation-desulfurization chemistry. Organic Letters 17(19), 49024905.

S. H. Scheres (2012). A Bayesian view on cryo-EM structure determination. Journal of Molecular Biology 415(2), 406418.

T. L. Schlick , Z. Ding , E. W. Kovacs & M. B. Francis (2005). Dual-surface modification of the tobacco mosaic virus. Journal of the American Chemical Society 127(11), 37183723.

M. J. Schmidt , A. Fedoseev , D. Bucker , J. Borbas , C. Peter , M. Drescher & D. Summerer (2015). EPR distance measurements in native proteins with genetically encoded spin labels. ACS Chemical Biology 10(12), 27642771.

M. J. Schmidt & D. Summerer (2013). Red-light-controlled protein-RNA crosslinking with a genetically encoded furan. Angewandte Chemie (International edition in English) 52(17), 46904693.

T. Schubeis , P. Yuan , M. Ahmed , M. Nagaraj , B. J. Van Rossum & C. Ritter (2015). Untangling a repetitive amyloid sequence: correlating biofilm-derived and segmentally labeled curli fimbriae by solid-state NMR spectroscopy. Angewandte Chemie (International edition in English) 54(49), 1466914672.

K. C. Schultz , L. Supekova , Y. Ryu , J. Xie , R. Perera & P. G. Schultz (2006). A genetically encoded infrared probe. Journal of the American Chemical Society 128(43), 1398413985.

E. C. Schwartz , L. Saez , M. W. Young & T. W. Muir (2007). Post-translational enzyme activation in an animal via optimized conditional protein splicing. Nature Chemical Biology 3(1), 5054.

C. P. Scott , E. Abel-santos , M. Wall , D. C. Wahnon & S. J. Benkovic (1999). Production of cyclic peptides and proteins in vivo . Proceedings of the National Academy of Sciences United States of America 96(24), 1363813643.

K. L. Seim , A. C. Obermeyer & M. B. Francis (2011). Oxidative modification of native protein residues using cerium(IV) ammonium nitrate. Journal of the American Chemical Society 133(42), 1697016976.

J. L. Seitchik , J. C. Peeler , M. T. Taylor , M. L. Blackman , T. W. Rhoads , R. B. Cooley , C. refakis , J. M. Fox & R. A. Mehl (2012). Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes. Journal of the American Chemical Society 134(6), 28982901.

R. Serwa , I. Wilkening , G. Del Signore , M. Muhlberg , I. Claussnitzer , C. Weise , M. Gerrits & C. P. Hackenberger (2009). Chemoselective Staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angewandte Chemie (International edition in English) 48(44), 82348239.

L. Shah , S. T. Laughlin & I. S. Carrico (2016). Light-activated staudinger-bertozzi ligation within living animals. Journal of the American Chemical Society 138(16), 51865189.

N. H. Shah , G. P. Dann , M. Vila-Perello , Z. Liu & T. W. Muir (2012). Ultrafast protein splicing is common among cyanobacterial split inteins: implications for protein engineering. Journal of the American Chemical Society 134(28), 1133811341.

N. H. Shah , E. Eryilmaz , D. Cowburn & T. W. Muir (2013). Naturally split inteins assemble through a ‘capture and collapse’ mechanism. Journal of the American Chemical Society 135(49), 1867318681.

N. H. Shah & T. W. Muir (2014). Inteins: nature's gift to protein chemists. Chemical Science 5(1), 446461.

N. H. Shah , M. Vila-Perello & T. W. Muir (2011). Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split intein. Angewandte Chemie (International edition in English) 50(29), 65116515.

S. Y. Shang , Z. P. Tan , S. W. Dong & S. J. Danishefsky (2011). An advance in proline ligation. Journal of the American Chemical Society 133(28), 1078410786.

N. G. Sharaf & A. M. Gronenborn (2015). (19)F-modified proteins and (19)F-containing ligands as tools in solution NMR studies of protein interactions. Methods in Enzymology 565, 6795.

J. Shi & T. W. Muir (2005). Development of a tandem protein trans-splicing system based on native and engineered split inteins. Journal of the American Chemical Society 127(17), 61986206.

P. Shieh & C. R. Bertozzi (2014). Design strategies for bioorthogonal smart probes. Organic & Biomolecular Chemistry 12(46), 93079320.

N. R. Silvaggi , L. J. Martin , H. Schwalbe , B. Imperiali & K. N. Allen (2007). Double-lanthanide-binding tags for macromolecular crystallographic structure determination. Journal of the American Chemical Society 129(22), 71147120.

P. Siman , S. V. Karthikeyan & A. Brik (2012). Native chemical ligation at glutamine. Organic Letters 14(6), 15201523.

M. D. Simon , F. Chu , L. R. Racki , C. C. De La Cruz , A. L. Burlingame , B. Panning , G. J. Narlikar & K. M. Shokat (2007). The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128(5), 10031012.

L. Skrisovska , M. Schubert & F. H. Allain (2010). Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins. Journal of Biomolecular NMR 46(1), 5165.

S. A. Slavoff , D. S. Liu , J. D. Cohen & A. Y. Ting (2011). Imaging protein–protein interactions inside living cells via interaction-dependent fluorophore ligation. Journal of the American Chemical Society 133(49), 1976919776.

A. H. Smits , A. Borrmann , M. Roosjen , J. C. Van Hest & M. Vermeulen (2016). Click-MS: tagless protein enrichment using bioorthogonal chemistry for quantitative proteomics. ACS Chemical Biology 11(12), 32453250.

F. Song , P. Chen , D. Sun , M. Wang , L. Dong , D. Liang , R. M. Xu , P. Zhu & G. Li (2014). Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344(6182), 376380.

C. D. Spicer & B. G. Davis (2014). Selective chemical protein modification. Nature Communications 5, 4740.

G. Srinivasan , C. M. James & J. A. Krzycki (2002). Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296(5572), 14591462.

N. Stephanopoulos & M. B. Francis (2011). Choosing an effective protein bioconjugation strategy. Nature Chemical Biology 7(12), 876884.

A. J. Stevens , Z. Z. Brown , N. H. Shah , G. Sekar , D. Cowburn & T. W. Muir (2016). Design of a split intein with exceptional protein splicing activity. Journal of the American Chemical Society 138(7), 21622165.

D. Summerer , S. Chen , N. Wu , A. Deiters , J. W. Chin & P. G. Schultz (2006). A genetically encoded fluorescent amino acid. Proceedings of the National Academy of Sciences United States of America 103(26), 97859789.

W. Szymanski , B. Wu , C. Poloni , D. B. Janssen & B. L. Feringa (2013). Azobenzene photoswitches for Staudinger-Bertozzi ligation. Angewandte Chemie (International edition in English) 52(7), 20682072.

Z. P. Tan , S. Y. Shang & S. J. Danishefsky (2010). Insights into the finer issues of native chemical ligation: an approach to cascade ligations. Angewandte Chemie (International edition in English) 49(49), 95009503.

K. Tanaka , M. Kitadani & K. Fukase (2011). Target-selective fluorescent ‘switch-on’ protein labeling by 6pi-azaelectrocyclization. Organic & Biomolecular 9(15), 53465349.

J. Tenboer , S. Basu , N. Zatsepin , K. Pande , D. Milathianaki , M. Frank , M. Hunter , S. Boutet , G. J. Williams , J. E. Koglin , D. Oberthuer , M. Heymann , C. Kupitz , C. Conrad , J. Coe , S. Roy-Chowdhury , U. Weierstall , D. James , D. Wang , T. Grant , A. Barty , O. Yefanov , J. Scales , C. Gati , C. Seuring , V. Srajer , R. Henning , P. Schwander , R. Fromme , A. Ourmazd , K. Moffat , J. J. Van Thor , J. C. Spence , P. Fromme , H. N. Chapman & M. Schmidt (2014). Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein. Science 346(6214), 12421246.

L. H. Tey , E. J. Loveridge , R. S. Swanwick , S. L. Flitsch & R. K. Allemann (2010). Highly site-selective stability increases by glycosylation of dihydrofolate reductase. FEBS Journal 277(9), 21712179.

C. S. Theile , M. D. Witte , A. E. Blom , L. Kundrat , H. L. Ploegh & C. P. Guimaraes (2013). Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nature Protocols 8(9), 18001807.

I. V. Thiel , G. Volkmann , S. Pietrokovski & H. D. Mootz (2014). An atypical naturally split intein engineered for highly efficient protein labeling. Angewandte Chemie (International edition in English) 53(5), 13061310.

R. E. Thompson , B. Chan , L. Radom , K. A. Jolliffe & R. J. Payne (2013). Chemoselective peptide ligation-desulfurization at aspartate. Angewandte Chemie (International edition in English) 52(37), 97239727.

R. E. Thompson , X. Liu , N. Alonso-Garcia , P. J. Pereira , K. A. Jolliffe & R. J. Payne (2014). Trifluoroethanethiol: an additive for efficient one-pot peptide ligation-desulfurization chemistry. Journal of the American Chemical Society 136(23), 81618164.

F. Tian , Y. Lu , A. Manibusan , A. Sellers , H. Tran , Y. Sun , T. Phuong , R. Barnett , B. Hehli , F. Song , M. J. Deguzman , S. Ensari , J. K. Pinkstaff , L. M. Sullivan , S. L. Biroc , H. Cho , P. G. Schultz , J. Dijoseph , M. Dougher , D. Ma , R. Dushin , M. Leal , L. Tchistiakova , E. Feyfant , H. P. Gerber & P. Sapra (2014). A general approach to site-specific antibody drug conjugates. Proceedings of the National Academy of Sciences United States of America 111(5), 17661771.

S. D. Tilley & M. B. Francis (2006). Tyrosine-selective protein alkylation using pi-allylpalladium complexes. Journal of the American Chemical Society 128(4), 10801081.

P. Tompa (2012). Intrinsically disordered proteins: a 10-year recap. Trends in Biochemical Sciences 37(12), 509516.

H. Ton-That , S. K. Mazmanian , K. F. Faull & O. Schneewind (2000). Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates. Journal of Biological Chemistry 275(13), 98769881.

N. I. Topilina & K. V. Mills (2014). Recent advances in in vivo applications of intein-mediated protein splicing. Mobile DNA 5(1), 5.

V. Y. Torbeev & D. Hilvert (2013). Both the cis-trans equilibrium and isomerization dynamics of a single proline amide modulate beta2-microglobulin amyloid assembly. Proceedings of the National Academy of Sciences United States of America 110(50), 2005120056.

V. Y. Torbeev , H. Raghuraman , D. Hamelberg , M. Tonelli , W. M. Westler , E. Perozo & S. B. Kent (2011). Protein conformational dynamics in the mechanism of HIV-1 protease catalysis. Proceedings of the National Academy of Sciences United States of America 108(52), 2098220987.

C. W. Tornoe , C. Christensen & M. Meldal (2002). Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. Journal of Organic Chemistry 67(9), 30573064.

M. L. Tremblay , L. Xu , T. Lefevre , M. Sarker , K. E. Orrell , J. Leclerc , Q. Meng , M. Pezolet , M. Auger , X. Q. Liu & J. K. Rainey (2015). Spider wrapping silk fibre architecture arising from its modular soluble protein precursor. Science Report 5, 11502.

V. Tugarinov , P. M. Hwang , J. E. Ollerenshaw & L. E. Kay (2003). Cross-correlated relaxation enhanced 1H[bond]13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. Journal of the American Chemical Society 125(34), 1042010428.