Mapping transient partial unfolding by protein engineering and native-state proteolysis.


Transient partial unfolding of proteins under native conditions may have significant consequences in the biochemical and biophysical properties of proteins. Native-state proteolysis offers a facile way to investigate the thermodynamic and kinetic accessibilities of partially unfolded forms (cleavable forms) under native conditions. However, determination of the structure of the cleavable form, which is populated only transiently, remains challenging. Although in some cases partially cleaved products from proteolysis provide information on the structure of this elusive form, proteolysis of many proteins does not accumulate detectable intermediates. Here, we describe a systematic approach to determining structures of cleavable forms by protein engineering and native-state proteolysis. By devising phi(c) analysis, which is analogous to conventional phi analysis, we have determined the structure of the cleavable form of Escherichia coli maltose-binding protein (MBP), which does not accumulate any partially cleaved products. We mutated 10 buried residues in MBP to alanine and determined phi(c) values from the effects of the mutations on global stability and proteolytic susceptibility. The result of this analysis suggests that two C-terminal helices in MBP are unfolded in their cleavable form. The effect of ligand binding on proteolytic susceptibility and C-terminal deletion mutations also confirms the proposed structure. Our approach and methodology are generally applicable not only in elucidating the mechanism of proteolysis but also in investigating other important processes involving partial unfolding under native conditions such as protein misfolding and aggregation. Study holds ProTherm entries: 24308, 24309, 24310, 24311, 24312, 24313, 24314, 24315, 24316, 24317, 24318, 24319, 24320, 24321, 24322, 24323, 24324, 24325, 24326, 24327, 24328, 24329, 24330, 24331, 24332, 24333, 24334, 24335, 24336, 24337, 24338, 24339 Extra Details: The Cm values were determined by fitting the change in ellipticity in varying concentrations of urea to a two-state equilibrium unfolding model. proteolysis; energy landscape; partial unfolding; mutagenesis; protein engineering

Submission Details

ID: Ph5sp9u23

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:55 p.m.

Version: 1

Publication Details
Chang Y;Park C,J. Mol. Biol. (2009) Mapping transient partial unfolding by protein engineering and native-state proteolysis. PMID:19683000
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 UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Maltose-binding periplasmic protein P0AEY0 MALE_ECO57
100.0 Maltose-binding periplasmic protein P0AEX9 MALE_ECOLI
94.3 Maltose-binding periplasmic protein P19576 MALE_SALTY
93.9 Maltose-binding periplasmic protein P18815 MALE_KLEAE
90.7 Maltose-binding periplasmic protein P41130 MALE_PHOLU