Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH.


Abstract

The stability of bovine lactoperoxidase to denaturation by guanidinium-HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near Tm is controlled by kinetics. The values for the thermal transition, Tm, at slow heating scan rate were 66.8, 61.1, and 47.2 degrees C in the presence of 0.5, 1, and 2 M guanidinium-HCl, respectively. The extrapolated value for Tm in the absence of guanidinium-HCl is 73.7 degrees C, compared with 70.2 degrees C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, Cp, at Tm and Ea for the thermal transition decrease under conditions where Tm is lowered. At a given concentration, urea is less effective than guanidinium-HCl in reducing Tm, but urea reduces Cp relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 degrees C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164-1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change. Study holds ProTherm entries: 25720, 25721, 25722, 25723, 25724, 25725, 25726, 25727, 25728, 25729, 25730, 25731, 25732, 25733, 25734, 25735, 25736, 25737, 25738, 25739, 25740, 25741, 25742 Extra Details: Scan rate: 0.25 degrees/min Protein stability; Denaturation

Submission Details

ID: wCVpmdKo

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:56 p.m.

Version: 1

Publication Details
Zelent B;Sharp KA;Vanderkooi JM,Biochim. Biophys. Acta (2010) Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH. PMID:20298816
Additional Information

Study Summary

Number of data points 43
Proteins Lactoperoxidase ; Lactoperoxidase
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp pH:8.9, details:Additives 1M GdnHCl ; Experimental Assay: Tm pH:8.9, details:Additives 1M GdnHCl ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:8.0 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:8.0 ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:7.0 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:7.0 ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:5.1 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:5.1 ; Experimental Assay: dCp details:Additives 1M GdnHCl, pH:4.1 ; Experimental Assay: Tm details:Additives 1M GdnHCl, pH:4.1 ; Experimental Assay: dCp pH:6.0, details:Additives 2M urea ; Experimental Assay: Tm pH:6.0, details:Additives 2M urea ; Experimental Assay: dCp pH:6.0, details:Additives 1M urea ; Experimental Assay: Tm pH:6.0, details:Additives 1M urea ; Experimental Assay: dCp pH:6.0, details:Additives 2M GdnHCl ; Experimental Assay: Tm pH:6.0, details:Additives 2M GdnHCl ; Experimental Assay: dCp pH:6.0, details:Additives 1M GdnHCl ; Experimental Assay: Tm pH:6.0, details:Additives 1M GdnHCl ; Experimental Assay: dCp pH:6.0, details:Additives 0.5M GdnHCl ; Experimental Assay: Tm pH:6.0, details:Additives 0.5M GdnHCl ; Experimental Assay: dCp pH:6.0, details:Additives ; Experimental Assay: Tm pH:6.0, details:Additives
Libraries Mutations for sequence VKCDENSPYRTITGDCNNRRSPALGAANRALARWLPAEYEDGLALPFGWTQRKTRNGFRVPLAREVSNKIVGYLDEEGVLDQNRSLLFMQWGQIVDHDLDFAPETELGSNEHSKTQCEEYCIQGDNCFPIMFPKNDPKLKTQGKCMPFFRAGFVCPTPPYQSLAREQINAVTSFLDASLVYGSEPSLASRLRNLSSPLGLMAVNQEAWDHGLAYLPFNNKKPSPCEFINTTARVPCFLAGDFRASEQILLATAHTLLLREHNRLARELKKLNPHWNGEKLYQEARKILGAFIQIITFRDYLPIVLGSEMQKWIPPYQGYNNSVDPRISNVFTFAFRFGHMEVPSTVSRLDENYQPWGPEAELPLHTLFFNTWRIIKDGGIDPLVRGLLAKKSKLMNQDKMVTSELRNKLFQPTHKIHGFDLAAINLQRCRDHGMPGYNSWRGFCGLSQPKTLKGLQTVLKNKILAKKLMDLYKTPDNIDIWIGGNAEPMVERGRVGPLLACLLGRQFQQIRDGDRFWWENPGVFTEKQRDSLQKVSFSRLICDNTHITKVPLHAFQANNYPHDFVDCSTVDKLDLSPWASREN

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
3Q9K 2011-03-02 1.7 Crystal structure of bovine lactoperoxidase complexed with Phenyl isothiocyanate at 1.7 A resolution
3QL6 2011-03-02 1.7 Crystal structure of the complex of bovine lactoperoxidase with nimesulide at 1.7 A resolution
3NYH 2010-10-20 1.77 Crystal structure of lactoperoxidase complexed simultaneously with thiocyanate ion, iodide ion, bromide ion, chloride ion through the substrate diffusion channel reveals a preferential queue of the inorganic substrates towards the distal heme cavity
5ZWW 2018-06-13 1.77 CRYSTAL STRUCTURE OF THE COMPLEX OF LACTOPEROXIDASE WITH HYDROGEN PEROXIDE AT 1.77A RESOLUTION
3OGW 2010-12-01 1.89 Structure of the complex of bovine lactoperoxidase with indomethacin at 1.9A resolution
6A4Y 2018-07-04 1.92 Crystal structure of bovine lactoperoxidase with partial occupancies of iodide and SCN- ions at the substrate binding site on the distal heme side at 1.92 A resolution
5GLS 2016-07-27 1.93 Structure of bovine Lactoperoxidase with a partially modified covalent bond with heme moiety
5B72 2016-07-13 1.98 Crystal structure of bovine lactoperoxidase with a broken covalent bond between Glu258 and heme moiety at 1.98 A resolution.
4KSZ 2013-07-10 1.98 Crystal structure of bovine lactoperoxidase complexed with cystiene at 1.98A resolution
4NT3 2013-12-25 1.99 Structure of the complex of bovine lactoperoxidase with 3,3'-dipyridyl ketone at 1.99 A resolution
3S4F 2011-06-08 2.0 Crystal structure of the complex of bovine lactoperoxidase with 1H-pyrazolo[4,3-c] pyridine at 1.99 A resolution
3V6Q 2012-02-15 2.0 Crystal structure of the complex of bovine lactoperoxidase with Carbon monoxide at 2.0 A resolution
3R4X 2011-08-03 2.01 Crystal structure of bovine lactoperoxidase complexed with pyrazine-2-carboxamide at 2 A resolution
5WV3 2017-02-15 2.07 Crystal structure of bovine lactoperoxidase with a partial Glu258-heme linkage at 2.07 A resolution.
4Y55 2015-03-25 2.1 Crystal structure of Buffalo lactoperoxidase with Rhodanide at 2.09 Angstrom resolution
5ZGS 2018-05-23 2.2 Crystal structure of the complex of bovine lactoperoxidase with multiple SCN and OSCN ions in the distal heme cavity
3KRQ 2010-05-26 2.25 Crystal structure of the complex of lactoperoxidase with a potent inhibitor amino-triazole at 2.2a resolution
3BXI 2008-03-25 2.3 Structure of the complex of bovine lactoperoxidase with its catalyzed product hypothiocyanate ion at 2.3A resolution
5GH0 2016-06-29 2.3 Crystal structure of the complex of bovine lactoperoxidase with mercaptoimidazole at 2.3 A resolution
4NJB 2013-11-27 2.31 Crystal structure of the complex of lactoperoxidase from bovine with 3,3-oxydipyridine at 2.31 A resolution
2QPK 2007-08-07 2.34 Crystal structure of the complex of bovine lactoperoxidase with salicylhydroxamic acid at 2.34 A resolution
3GCJ 2009-03-31 2.34 Mode of ligand binding and assignment of subsites in mammalian peroxidases: crystal structure of lactoperoxidase complexes with acetyl salycylic acid, salicylhydroxamic acid and benzylhydroxamic acid
2PT3 2007-05-22 2.34 Crystal structure of bovine lactoperoxidase at 2.34 A resolution reveals multiple anion binding sites
3TGY 2011-09-21 2.35 Crystal structure of the complex of Bovine Lactoperoxidase with Ascorbic acid at 2.35 A resolution
3ERH 2009-03-31 2.4 First structural evidence of substrate specificity in mammalian peroxidases: Crystal structures of substrate complexes with lactoperoxidases from two different species
4PNX 2014-03-12 2.41 Crystal structure of the complex of lactoperoxidase with bromo methane at 2.41 angstrom resolution
3PY4 2011-01-05 2.42 Crystal structure of bovine lactoperoxidase in complex with paracetamol at 2.4A resolution
4GN6 2012-09-19 2.42 Structure of paracetamol bound bovine lactoperoxidase at 2.45A resolution.
3FNL 2009-01-27 2.48 Crystal Structure of the Complex of Buffalo Lactoperoxidase with Salicylhydroxamic Acid at 2.48 A Resolution
3GCL 2009-03-31 2.5 Mode of ligand binding and assignment of subsites in mammalian peroxidases: crystal structure of lactoperoxidase complexes with acetyl salycylic acid, salicylhydroxamic acid and benzylhydroxamic acid
2QRB 2007-08-14 2.5 Crystal structure of chloride saturated bovine lactoperoxidase at 2.5 A resolution shows multiple halide binding sites
3ERI 2009-03-31 2.5 First structural evidence of substrate specificity in mammalian peroxidases: Crystal structures of substrate complexes with lactoperoxidases from two different species
3GC1 2009-03-31 2.5 Crystal structure of bovine lactoperoxidase
2QQT 2007-08-07 2.5 Crystal structure of the complex of bovine lactoperoxidase with acetyl salicylic acid at 2.5 A resolution
3R5O 2011-08-17 2.6 Crystal structure of the complex of bovine lactoperoxidase with 4-allyl-2-methoxyphenol at 2.6 A resolution
4GM7 2012-09-19 2.6 Structure of cinnamic acid bound bovine lactoperoxidase at 2.6A resolution.
3UBA 2011-12-28 2.65 Crystal structure of the complex of bovine lactoperoxidase with p-hydroxycinnamic acid at 2.6 A resolution
3FAQ 2009-03-31 2.7 Crystal structure of lactoperoxidase complex with cyanide
2PUM 2007-05-22 2.7 Crystal structure of bovine lactoperoxidase complex with catechol and iodide at 2.7 A resolution
3I6N 2009-10-13 2.7 Mode of Binding of the Tuberculosis Prodrug Isoniazid to Peroxidases: Crystal Structure of Bovine Lactoperoxidase with Isoniazid at 2.7 Resolution
2GJM 2006-05-02 2.75 Crystal structure of Buffalo lactoperoxidase at 2.75A resolution
2O86 2006-12-26 2.8 Crystal structure of a ternary complex of buffalo lactoperoxidase with nitrate and iodide at 2.8 A resolution
3GCK 2009-03-31 2.9 Mode of ligand binding and assignment of subsites in mammalian peroxidases: crystal structure of lactoperoxidase complexes with acetyl salycylic acid, salicylhydroxamic acid and benzylhydroxamic acid
2NQX 2006-11-21 2.95 Crystal Structure of bovine lactoperoxidase with iodide ions at 2.9A resolution
2IPS 2006-10-24 3.1 Crystal structure of a ternary complex of bovine lactoperoxidase with thiocyanate and iodide at 3.1 A resolution
2Z5Z 2007-08-14 3.5 Crystal structure of the complex of buffalo Lactoperoxidase with fluoride ion at 3.5A resolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.7 Lactoperoxidase A5JUY8 PERL_BUBBU
100.0 Lactoperoxidase P80025 PERL_BOVIN