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

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 SKSKPDTSAPTSPKVTYKAPVPSGEVYFADSFDRGTLSGWILSKAKKDDTDDEIAKYDGKWEVDEMKETKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTKPLIVQYEVNFQNGIECGGAYVKLLSKTPELNLDQFHDKTPYTIMFGPDKCGEDYKLHFIFRHKNPKTGVYEEKHAKRPDADLKTYFTDKKTHLYTLILNPDNSFEILVDQSIVNSGNLLNDMTPPVNPSREIEDPEDQKPEDWDERPKIPDPDAVKPDDWNEDAPAKIPDEEATKPDGWLDDEPEYVPDPDAEKPEDWDEDMDGEWEAPQIANPKCESAPGCGVWQRPMIDNPNYKGKWKPPMIDNPNYQGIWKPRKIPNPDFFEDLEPFKMTPFSAIGLELWSMTSDIFFDNFIVCGDRRVVDDWANDGWGLKKAADGAAEP ; Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV ; Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL ; Mutations for sequence KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL
Sequence Assay Result Units