Use of fluorescence energy transfer to characterize the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding.


Abstract

The CL fragment of a type-kappa immunoglobulin light chain in which the C-terminal cysteine residue was modified with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (CL-AEDANS fragment) was prepared. This fragment has only one tryptophan residue at position 148. The compactness of the fragment whose intrachain disulfide bond was reduced in order for the tryptophan residue to fluoresce (reduced CL-AEDANS fragment) was studied in the early stages of refolding from 4 M guanidine hydrochloride by fluorescence energy transfer from Trp 148 to the AEDANS group. The AEDANS group attached to the SH group of a cysteine scarcely fluoresced when excited at 295 nm. For the reduced CL-AEDANS fragment, the fluorescence emission band of the Trp residue overlapped with the absorption band of the AEDANS group, and the fluorescence energy transfer was observed between Trp 148 and the AEDANS group in the absence of guanidine hydrochloride. In 4 M guanidine hydrochloride, the distance between the donor and the acceptor was larger, and the efficiency of the energy transfer became lower. The distance between Trp 148 and the AEDANS group for the intact protein estimated by using the energy-transfer data was in good agreement with that obtained by X-ray crystallographic analysis. By the use of fluorescence energy transfer, tryptophyl fluorescence, and circular dichroism at 218 nm, the kinetics of unfolding and refolding of the reduced fragment were studied. These three methods gave the same unfolding kinetic pattern. However, the refolding kinetics measured by fluorescence energy transfer were different from those measured by tryptophyl fluorescence and circular dichroism, the latter two giving the same kinetic pattern.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4380 Extra Details: constant fragment; compactness; energy transfer;,cis-trans isomerization

Submission Details

ID: inKcjFdD3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Kawata Y;Hamaguchi K,Biochemistry (1991) Use of fluorescence energy transfer to characterize the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding. PMID:1902379
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
2Q20 2008-04-08 1.3 Structure of the germline Vk1 O18/O8 light chain variable domain homodimer
1EEQ 2001-02-01 1.5 M4L/Y(27D)D/T94H Mutant of LEN
3CDF 2008-09-02 1.53 kI O18/O8 Y87H immunoglobulin light chain variable domain
3CDC 2008-09-02 1.53 kI O18/O8 N34I/Y87H immunoglobulin light chain variable domain
1EEU 2001-02-03 1.6 M4L/Y(27D)D/Q89D/T94H mutant of LEN
1EFQ 2001-02-09 1.6 Q38D mutant of LEN
4L1H 2014-06-04 1.68 Bence-Jones immunoglobulin REI variable portion with seven point mutations
1BWW 1998-10-07 1.7 BENCE-JONES IMMUNOGLOBULIN REI VARIABLE PORTION, T39K MUTANT
1B0W 1998-11-16 1.8 Structural comparison of amyloidogenic light chain dimer in two crystal forms with nonamyloidogenic counterparts
1QAC 2000-02-23 1.8 CHANGE IN DIMERIZATION MODE BY REMOVAL OF A SINGLE UNSATISFIED POLAR RESIDUE
1WTL 1994-11-01 1.9 COMPARISON OF CRYSTAL STRUCTURES OF TWO HOMOLOGOUS PROTEINS: STRUCTURAL ORIGIN OF ALTERED DOMAIN INTERACTIONS IN IMMUNOGLOBULIN LIGHT CHAIN DIMERS
1EK3 2001-03-06 1.9 KAPPA-4 IMMUNOGLOBULIN VL, REC
1LVE 1998-01-21 1.95 STRUCTURE OF THE VARIABLE DOMAIN OF HUMAN IMMUNOGLOBULIN K-4 LIGHT CHAIN LEN
1REI 1976-05-19 2.0 THE MOLECULAR STRUCTURE OF A DIMER COMPOSED OF THE VARIABLE PORTIONS OF THE BENCE-JONES PROTEIN REI REFINED AT 2.0 ANGSTROMS RESOLUTION
3LVE 1999-05-18 2.0 LEN Q38E MUTANT: A DOMAIN FLIP FROM A SINGLE AMINO ACID SUBSTITUTION
5LVE 2000-02-18 2.0 STRUCTURE OF THE VARIABLE DOMAIN OF HUMAN IMMUNOGLOBULIN K-4 LIGHT CHAIN LEN
1QP1 1999-06-17 2.06 KAPPA VARIABLE LIGHT CHAIN
1JV5 2002-01-09 2.2 Anti-blood group A Fv
4LVE 1999-05-18 2.3 LEN K30T MUTANT: A DOMAIN FLIP AS A RESULT OF A SINGLE AMINO ACID SUBSTITUTION
3CDY 2008-09-02 2.43 AL-09 H87Y, immunoglobulin light chain variable domain
2LVE 1999-05-18 2.7 RECOMBINANT LEN
1AR2 1997-11-12 2.8 DISULFIDE-FREE IMMUNOGLOBULIN FRAGMENT
4K07 2013-10-30 2.83 Crystal structure of the amyloid-forming immunoglobulin AL-103 cis-proline 95 mutant
5XP1 2017-08-02 2.88 Structure of monomeric mutant of REI immunoglobulin light chain variable domain crystallized at pH 6

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.0 Immunoglobulin kappa variable 4-1 P06312 KV401_HUMAN
100.0 Immunoglobulin kappa variable 1D-33 P01593 KVD33_HUMAN
100.0 Immunoglobulin kappa variable 1D-33 P01594 KV133_HUMAN