Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme.


Abstract

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability. Study holds ProTherm entries: 7916, 7917 Extra Details: N->I cooperative stages; intermediate; ring-current shifts;,aromatic cluster

Submission Details

ID: JCyuxVRb

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Koshiba T;Yao M;Kobashigawa Y;Demura M;Nakagawa A;Tanaka I;Kuwajima K;Nitta K,Biochemistry (2000) Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme. PMID:10727216
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
1I56 2002-02-27 SOLUTION STRUCTURE OF CA2+-BOUND STATE OF CANINE MILK LYSOZYME
1QQY 2000-06-09 1.85 X-RAY CRYSTAL STRUCTURE ANALYSIS OF CANINE MILK LYSOZYME (APO-TYPE)
1EL1 2001-03-13 1.9 X-RAY CRYSTAL STRUCTURE ANALYSIS OF CANINE MILK LYSOZYME (HOLO-TYPE)
2CWI 2006-06-20 1.94 X-ray crystal structure analysis of recombinant wild-type canine milk lysozyme (apo-type)
2Z2E 2007-11-27 2.01 Crystal Structure of Canine Milk Lysozyme Stabilized against Non-enzymatic Deamidation

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lysozyme C, milk isozyme P81708 LYSC1_CANLF