A mass spectrometry-based probe of equilibrium intermediates in protein-folding reactions.


Abstract

Described here is a mass spectrometry- and H/D exchange-based approach for the detection of equilibrium intermediate state(s) in protein-folding reactions. The approach utilizes the stability of unpurified proteins from rates of H/D exchange (SUPREX) technique to measure the m value (i.e., delta DeltaG/delta [denaturant] value) associated with the folding reaction of a protein. Such SUPREX m-value analyses can be made over a wide range of denaturant concentrations. Thus, the described approach is well-suited for the detection of high-energy intermediates that might be populated at low denaturant concentrations and hard to detect in conventional chemical denaturation experiments using spectroscopic probes. The approach is demonstrated on four known non-two-state folding proteins, including alpha-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin. The non-two-state folding behavior of each model protein system was detected by the described method. The cytochrome c, myoglobin, and IFABP systems each had high-energy intermediate states that were undetected in conventional optical spectroscopy-based studies and previously required other more specialized biophysical approaches (e.g., nuclear magnetic resonance spectroscopy-based methods and protease protection assays) for their detection. The SUPREX-based approach outlined here offers an attractive alternative to these other approaches, because it has the advantage of speed and the ability to analyze both purified and unpurified protein samples in either concentrated or dilute solution. Study holds ProTherm entries: 22067, 22068, 22069, 22070, 22071, 22072, 22073, 22074, 22075, 22076 Extra Details: equilibrium intermediate state, high-energy intermediates, R-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin.

Submission Details

ID: fY5e4x6d

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:53 p.m.

Version: 1

Publication Details
Dai SY;Fitzgerald MC,Biochemistry (2006) A mass spectrometry-based probe of equilibrium intermediates in protein-folding reactions. PMID:17042507
Additional Information

Study Summary

Number of data points 14
Proteins Cytochrome c ; Fatty acid-binding protein, intestinal ; Alpha-lactalbumin ; Cytochrome c ; Myoglobin ; Alpha-lactalbumin ; Myoglobin ; Fatty acid-binding protein, intestinal
Unique complexes 4
Assays/Quantities/Protocols Experimental Assay: m pH:5.8, ionic:KCl: 20 mM, buffers:Sodium acetate: 20 mM ; Experimental Assay: dG_H2O pH:5.8, ionic:KCl: 20 mM, buffers:Sodium acetate: 20 mM ; Experimental Assay: m pH:7.5, ionic:KCl: 100 mM, buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM ; Experimental Assay: dG_H2O pH:7.5, ionic:KCl: 100 mM, buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM ; Experimental Assay: m buffers:Tris: 20 mM, pH:7.4, ionic:: ; Experimental Assay: dG_H2O buffers:Tris: 20 mM, pH:7.4, ionic:: ; Experimental Assay: m pH:7.5, ionic:KCl: 100 mM, buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM ; Experimental Assay: m pH:7.4, ionic:: , buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM
Libraries Mutations for sequence GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG ; Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL ; Mutations for sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE ; Mutations for sequence AFDGTWKVDRNENYEKFMEKMGINVVKRKLGAHDNLKLTITQEGNKFTVKESSNFRNIDVVFELGVDFAYSLADGTELTGTWTMEGNKLVGKFKRVDNGKELIAVREISGNELIQTYTYEGVEAKRIFKKE

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
1KTD 2002-01-15T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF CLASS II MHC MOLECULE IEK BOUND TO PIGEON CYTOCHROME C PEPTIDE
5YCJ 2017-09-07T00:00:00+0000 1.58 Ancestral myoglobin aMbWb' of Basilosaurus relative (polyphyly) imidazole-ligand
5YCI 2017-09-07T00:00:00+0000 1.97 Ancestral myoglobin aMbWb' of Basilosaurus relative (polyphyly)
5DF5 2015-08-26T00:00:00+0000 1.3 The structure of oxidized rat cytochrome c (T28E) at 1.30 angstroms resolution.
5C0Z 2015-06-12T00:00:00+0000 1.12 The structure of oxidized rat cytochrome c at 1.13 angstroms resolution
6N1O 2018-11-09T00:00:00+0000 1.55 Oxidized rat cytochrome c mutant (S47E)
5C9M 2015-06-28T00:00:00+0000 1.36 The structure of oxidized rat cytochrome c (T28A) at 1.362 angstroms resolution.
1HMK 1998-11-26T00:00:00+0000 2.0 RECOMBINANT GOAT ALPHA-LACTALBUMIN
6FF5 2018-01-03T00:00:00+0000 1.74 X-ray structure of bovine heart cytochrome c at high ionic strength
3J2T 2012-12-23T00:00:00+0000 9.5 An improved model of the human apoptosome

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.3 Cytochrome c B4USV4 CYC_OTOGA
91.3 Cytochrome c P00021 CYC_COLLI
92.2 Cytochrome c P00020 CYC_ANAPL
92.3 Cytochrome c P81280 CYC_ALLMI
90.5 Cytochrome c Q52V10 CYC_SAISC
93.3 Cytochrome c P00012 CYC_MIRLE
93.3 Cytochrome c Q52V09 CYC_CEPBA
93.3 Cytochrome c P00013 CYC_MINSC
93.3 Cytochrome c P00014 CYC_MACGI
94.3 Cytochrome c P00011 CYC_CANLF
94.3 Cytochrome c P62898 CYC_RAT
94.3 Cytochrome c P00008 CYC_RABIT
94.3 Cytochrome c P62897 CYC_MOUSE
95.2 Cytochrome c P68098 CYC_LAMGU
95.2 Cytochrome c P68100 CYC_ESCRO
95.2 Cytochrome c P68099 CYC_CAMDR
94.3 Cytochrome c P00007 CYC_HIPAM
97.1 Cytochrome c P62896 CYC_SHEEP
97.1 Cytochrome c P62895 CYC_PIG
97.1 Cytochrome c P62894 CYC_BOVIN
99.0 Cytochrome c P68096 CYC_EQUBU
99.0 Cytochrome c P68097 CYC_EQUAS
100.0 Cytochrome c P00004 CYC_HORSE
92.4 Fatty acid-binding protein, intestinal P55050 FABPI_MOUSE
100.0 Fatty acid-binding protein, intestinal P02693 FABPI_RAT
95.1 Alpha-lactalbumin P00712 LALBA_CAPHI
97.2 Alpha-lactalbumin P09462 LALBA_SHEEP
98.6 Alpha-lactalbumin Q9TSN6 LALBA_BUBBU
99.3 Alpha-lactalbumin Q9TSR4 LALBA_BOSMU
100.0 Alpha-lactalbumin P00711 LALBA_BOVIN
90.3 Myoglobin P02163 MYG_ROUAE
90.9 Myoglobin P11343 MYG_LUTLU
90.3 Myoglobin P02165 MYG_TUPGL
90.3 Myoglobin P02167 MYG_NYCCO
90.3 Myoglobin Q0KIY0 MYG_MESST
90.3 Myoglobin P02183 MYG_MESCA
90.8 Myoglobin P02177 MYG_ESCRO
90.8 Myoglobin Q0KIY2 MYG_BALED
90.8 Myoglobin Q0KIY1 MYG_BALBO
90.9 Myoglobin P02189 MYG_PIG
90.9 Myoglobin P02166 MYG_PERPO
92.2 Myoglobin P02169 MYG_LEPMU
91.6 Myoglobin P02181 MYG_INIGE
100.0 Myoglobin P68082 MYG_HORSE
100.0 Myoglobin P68083 MYG_EQUBU
90.1 Myoglobin P02178 MYG_MEGNO