Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification.


Abstract

An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence recognition tag for transglutaminase (TGase) affords stoichiometrically D-/A-labeled protein suitable for single-molecule FRET experiments. Thermodynamic data indicate that neither the presence of the TGase tag nor D/A labeling perturbs protein stability. As the N terminus in proteins is typically solvent accessible, a TGase tag can (in principle) be appended to any protein of interest by genetic engineering. Two-step chemical/enzymatic labeling may thus represent a simple, low-cost, and widely available strategy for D/A labeling of proteins for FRET-based single-molecule protein folding studies, even for non-protein-experts laboratories. Study holds ProTherm entries: 20569, 20570, 20571 Extra Details: protein labeling; transglutaminase; fluorescence resonance energy transfer; single-molecule spectroscopy; alternating laser excitation; fluorescence-aided molecular sorting

Submission Details

ID: taoygauq

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
J├Ąger M;Nir E;Weiss S,Protein Sci. (2006) Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification. PMID:16452617
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3CI2 1993-10-31 REFINEMENT OF THE THREE-DIMENSIONAL SOLUTION STRUCTURE OF BARLEY SERINE PROTEINASE INHIBITOR 2 AND COMPARISON WITH THE STRUCTURES IN CRYSTALS
1CIR 1996-01-29 COMPLEX OF TWO FRAGMENTS OF CI2 [(1-40)(DOT)(41-64)]
1CIS 1993-10-31 CONTEXT DEPENDENCE OF PROTEIN SECONDARY STRUCTURE FORMATION. THE THREE-DIMENSIONAL STRUCTURE AND STABILITY OF A HYBRID BETWEEN CHYMOTRYPSIN INHIBITOR 2 AND HELIX E FROM SUBTILISIN CARLSBERG
1LW6 2002-08-21 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1YPC 1994-01-31 1.7 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
5FFN 2016-05-18 1.8 Complex of subtilase SubTY from Bacillus sp. TY145 with chymotrypsin inhibitor CI2A
1CQ4 1998-11-25 1.8 CI2 MUTANT WITH TETRAGLUTAMINE (MGQQQQGM) REPLACING MET59
5FBZ 2016-05-18 1.9 Structure of subtilase SubHal from Bacillus halmapalus - complex with chymotrypsin inhibitor CI2A
2CI2 1988-09-07 2.0 CRYSTAL AND MOLECULAR STRUCTURE OF THE SERINE PROTEINASE INHIBITOR CI-2 FROM BARLEY SEEDS
1YPB 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
1YPA 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
2SNI 1988-09-07 2.1 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
1COA 1994-01-31 2.2 THE EFFECT OF CAVITY CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR 2
1CIQ 1996-03-08 2.2 COMPLEX OF TWO FRAGMENTS OF CI2, RESIDUES 1-40 AND 41-64

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.6 Chymotrypsin inhibitor 2 P08626 ICI3_HORVU
98.8 Subtilisin-chymotrypsin inhibitor-2A P01053 ICI2_HORVU