A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea).


Abstract

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation. Study holds ProTherm entries: 23935, 23936, 23937, 23938, 23939, 23940, 23941, 23942, 23943, 23944, 23945, 23946, 23947, 23948, 23949, 23950, 23951, 23952, 23953, 23954, 23955, 23956, 23957, 23958, 23959, 23960, 23961, 23962, 23963, 23964, 23965, 23966, 23967, 23968, 23969, 23970, 23971, 23972, 23973, 23974, 23975, 23976 Extra Details: Protein folding; Protein denaturation; Heat capacity

Submission Details

ID: FSZxuimv3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Singh R;Ali Dar T;Ahmad S;Moosavi-Movahedi AA;Ahmad F,Biophys. Chem. (2008) A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea). PMID:18201812
Additional Information

Study Summary

Number of data points 84
Proteins Ribonuclease pancreatic ; Chymotrypsinogen A ; Alpha-lactalbumin ; Lysozyme C ; Ribonuclease pancreatic ; Lysozyme C ; Alpha-lactalbumin ; Calnexin
Unique complexes 4
Assays/Quantities/Protocols Experimental Assay: dCp pH:2.0 ; Experimental Assay: Tm pH:2.0 ; Experimental Assay: dHvH pH:2.0 ; Experimental Assay: dG_H2O pH:2.0 ; Experimental Assay: dG_H2O ionic:NaCl: 0.1 M, buffers:Cacodylic acid: 0.05 M, pH:7.0 ; Experimental Assay: dG_H2O ionic:: , pH:2.0, buffers:KCl-HCl: 0.1 M ; Experimental Assay: dG_H2O pH:2.2 ; Experimental Assay: dCp ionic:NaCl: 0.1 M, buffers:Cacodylic acid: 0.05 M, pH:7.0 ; Experimental Assay: Tm ionic:NaCl: 0.1 M, buffers:Cacodylic acid: 0.05 M, pH:7.0 ; Experimental Assay: dHvH ionic:NaCl: 0.1 M, buffers:Cacodylic acid: 0.05 M, pH:7.0 ; Experimental Assay: dG_H2O ionic:NaCl: 0.1 M, buffers:Cacodylic acid: 0.05 M, pH:7.0 ; Experimental Assay: dCp ionic:: , pH:2.0, buffers:KCl-HCl: 0.1 M ; Experimental Assay: Tm ionic:: , pH:2.0, buffers:KCl-HCl: 0.1 M ; Experimental Assay: dHvH ionic:: , pH:2.0, buffers:KCl-HCl: 0.1 M ; Experimental Assay: dG_H2O ionic:: , pH:2.0, buffers:KCl-HCl: 0.1 M ; Experimental Assay: dCp pH:2.2 ; Experimental Assay: Tm pH:2.2 ; Experimental Assay: dHvH pH:2.2 ; Experimental Assay: dG_H2O pH:2.2
Libraries Mutations for sequence KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL ; Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL ; Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV ; Mutations for sequence SKSKPDTSAPTSPKVTYKAPVPSGEVYFADSFDRGTLSGWILSKAKKDDTDDEIAKYDGKWEVDEMKETKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTKPLIVQYEVNFQNGIECGGAYVKLLSKTPELNLDQFHDKTPYTIMFGPDKCGEDYKLHFIFRHKNPKTGVYEEKHAKRPDADLKTYFTDKKTHLYTLILNPDNSFEILVDQSIVNSGNLLNDMTPPVNPSREIEDPEDQKPEDWDERPKIPDPDAVKPDDWNEDAPAKIPDEEATKPDGWLDDEPEYVPDPDAEKPEDWDEDMDGEWEAPQIANPKCESAPGCGVWQRPMIDNPNYKGKWKPPMIDNPNYQGIWKPRKIPNPDFFEDLEPFKMTPFSAIGLELWSMTSDIFFDNFIVCGDRRVVDDWANDGWGLKKAADGAAEP

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
1JHN 2001-06-28T00:00:00+0000 2.9 Crystal Structure of the Lumenal Domain of Calnexin
2IHL 1993-06-29T00:00:00+0000 1.4 LYSOZYME (E.C.3.2.1.17) (JAPANESE QUAIL)
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
1FBI 1995-01-19T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF A CROSS-REACTION COMPLEX BETWEEN FAB F9.13.7 AND GUINEA-FOWL LYSOZYME
1GHL 1993-05-04T00:00:00+0000 2.1 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1HHL 1993-05-04T00:00:00+0000 1.9 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1JHL 1993-05-04T00:00:00+0000 2.4 THREE-DIMENSIONAL STRUCTURE OF A HETEROCLITIC ANTIGEN-ANTIBODY CROSS-REACTION COMPLEX
1BQL 1995-02-03T00:00:00+0000 2.6 STRUCTURE OF AN ANTI-HEL FAB FRAGMENT COMPLEXED WITH BOBWHITE QUAIL LYSOZYME
1DKJ 1996-01-10T00:00:00+0000 2.0 BOBWHITE QUAIL LYSOZYME
1DKK 1996-01-10T00:00:00+0000 1.9 BOBWHITE QUAIL LYSOZYME WITH NITRATE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
100.0 Chymotrypsinogen A P00766 CTRA_BOVIN
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
93.0 Lysozyme C P81711 LYSC_SYRSO
92.3 Lysozyme C P49663 LYSC_PHAVE
94.6 Lysozyme C P24533 LYSC_SYRRE
93.0 Lysozyme C P24364 LYSC_LOPLE
92.2 Lysozyme C P00704 LYSC_NUMME
95.3 Lysozyme C Q7LZT2 LYSC_TRATE
95.3 Lysozyme C P22910 LYSC_CHRAM
96.1 Lysozyme C P19849 LYSC_PAVCR
96.1 Lysozyme C Q7LZI3 LYSC_TRASA
96.9 Lysozyme C Q7LZP9 LYSC_LOPIM
96.9 Lysozyme C Q7LZQ0 LYSC_CATWA
96.9 Lysozyme C P00699 LYSC_CALCC
96.9 Lysozyme C P00700 LYSC_COLVI
93.2 Lysozyme C P00702 LYSC_PHACO
95.2 Lysozyme C P00703 LYSC_MELGA
95.3 Lysozyme C P00701 LYSC_COTJA
100.0 Lysozyme C P00698 LYSC_CHICK
96.4 Calnexin P35565 CALX_RAT
97.6 Calnexin P35564 CALX_MOUSE
96.1 Calnexin P27824 CALX_HUMAN
96.4 Calnexin Q5R440 CALX_PONAB
99.8 Calnexin P24643 CALX_CANLF