Molecular mechanism underlying the thermal stability and pH-induced unfolding of CHABII.


Abstract

The 37-residue alpha/beta protein CHABII was previously demonstrated to undergo a gradual pH-induced unfolding. It has been shown that even at pH 4.0 CHABII still retained a highly native-like secondary structure and tertiary topology although its tight side-chain packing was severely disrupted, typical of the molten globule state. Here, we have expressed and refolded the recombinant proteins of CHABII and its mutant [Phe21]-CHABII, and subsequently conducted extensive CD and NMR characterizations. The results indicated: (1) replacement of His21 by Phe in [Phe21]-CHABII eliminated the pH-induced unfolding from pH 6.5 to 4.0, indicating that His21 was responsible for the observed pH-induced unfolding of CHABII. Further examinations revealed that although the pH-induced unfolding of CHABII was also triggered by the protonation of the His residue as previously uncovered for apomyoglobin, their molecular mechanisms are different. (2) Monitoring the pH-induced unfolding by 1H-15N HSQC spectroscopy allowed us to visualize the gradual development of the CHABII molten globule. At pH 4.0, the HSQC spectrum of CHABII was poorly dispersed with dispersions of approximately 1 ppm over proton dimension and 10 ppm over 15N dimension, characteristic of severely or even "completely unfolded" proteins. One the other hand, unambiguous assignments of the NOESY spectra of CHABII led to the identification of the persistent medium and long-range NOEs at pH 4.0, which define a highly native-like secondary structure and tertiary packing. This implies that the degree of the native-like topology might be underestimated in the previous characterization of partially folded and even completely unfolded proteins. (3) Replacement of His21 by Phe with higher side-chain hydrophobicity only caused a minor structural rearrangement but considerably enhanced the packing interaction of the hydrophobic core, as evident from a dramatic increase in NOE contacts in [Phe21]-CHABII. The enhancement led to an increase of the thermal stability of [Phe21]-CHABII by approximately 17 deg. C. Study holds ProTherm entries: 18515 Extra Details: CHABII; protein folding; molten globule; thermal stability; NMR

Submission Details

ID: UV748nW43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Wei Z;Song J,J. Mol. Biol. (2005) Molecular mechanism underlying the thermal stability and pH-induced unfolding of CHABII. PMID:15808864
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
2CRD 1993-07-15 ANALYSIS OF SIDE-CHAIN ORGANIZATION ON A REFINED MODEL OF CHARYBDOTOXIN: STRUCTURAL AND FUNCTIONAL IMPLICATIONS
1CMR 1996-08-01 NMR SOLUTION STRUCTURE OF A CHIMERIC PROTEIN, DESIGNED BY TRANSFERRING A FUNCTIONAL SNAKE BETA-HAIRPIN INTO A SCORPION ALPHA/BETA SCAFFOLD (PH 3.5, 20C), NMR, 18 STRUCTURES
1BAH 1997-01-11 A TWO DISULFIDE DERIVATIVE OF CHARYBDOTOXIN WITH DISULFIDE 13-33 REPLACED BY TWO ALPHA-AMINOBUTYRIC ACIDS, NMR, 30 STRUCTURES
2A9H 2006-01-10 NMR structural studies of a potassium channel / charybdotoxin complex
4JTA 2013-06-12 2.5 Crystal structure of Kv1.2-2.1 paddle chimera channel in complex with Charybdotoxin
4JTD 2013-06-12 2.54 Crystal structure of Kv1.2-2.1 paddle chimera channel in complex with Lys27Met mutant of Charybdotoxin
4JTC 2013-06-12 2.56 Crystal structure of Kv1.2-2.1 paddle chimera channel in complex with Charybdotoxin in Cs+

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.9 Potassium channel toxin alpha-KTx 1.1 P59944 KAX1D_LEIHE
96.6 Potassium channel toxin alpha-KTx 1.1 P59943 KAX1C_LEIHE
100.0 Potassium channel toxin alpha-KTx 1.1 P13487 KAX11_LEIHE