Stability of proteins in the presence of carbohydrates; experiments and modeling using scaled particle theory.


Abstract

The effects of sucrose and fructose on the free energy of unfolding, DeltaG(N-->D), and on the change in hydrodynamic radius, R(H), upon unfolding were measured for RNase A and alpha-lactalbumin. Recently we analyzed the results for RNase A and showed that the effects of the carbohydrates on the protein's thermal stability can be accurately accounted for by scaled particle theory (SPT), and are thus largely entropic in nature. In this paper we extend this analysis to alpha-lactalbumin and demonstrate the generality of this finding. We also investigate the relationship between SPT and the thermodynamic formalism of preferential interactions. The preferential binding parameters calculated using SPT are in excellent agreement with experimentally measured values available in the literature. This agreement is expected to hold as long as enthalpic interactions between the cosolute and the protein are not important, as appears to be the case here. Finally we use the experimental data and SPT to calculate the change in the number of sugar molecules excluded from the protein surface during unfolding from knowledge of the preferential binding parameter for the native and denatured state of the protein. Study holds ProTherm entries: 22462, 22463, 22464, 22465, 22466, 22467, 22468, 22469, 22470, 22471, 22472, 22473, 22474, 22475, 22476, 22477, 22478, 22479, 22480, 22481, 22482, 22483, 22484, 22485, 22486, 22487, 22488, 22489, 22490, 22491, 22492, 22493, 22494, 22495, 22496, 22497, 22498, 22499, 22500, 22501, 22502, 22503 Extra Details: Protein stability; Carbohydrate; Scaled particle theory; Preferential interactions; Capillary electrophoresis

Submission Details

ID: ccNHnuTV4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:53 p.m.

Version: 1

Publication Details
O'Connor TF;Debenedetti PG;Carbeck JD,Biophys. Chem. (2007) Stability of proteins in the presence of carbohydrates; experiments and modeling using scaled particle theory. PMID:17234323
Additional Information

Study Summary

Number of data points 46
Proteins Ribonuclease pancreatic ; Ribonuclease pancreatic ; Alpha-lactalbumin ; Alpha-lactalbumin
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: dCp ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: Tm ionic:NaCl,CaCl2: 25 mM,35 microM, units:K ; Experimental Assay: dHvH ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: dCp ionic:NaCl: 30 mM ; Experimental Assay: Tm units:K, ionic:NaCl: 30 mM ; Experimental Assay: dHvH ionic:NaCl: 30 mM ; Experimental Assay: dG ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.83 M ; Experimental Assay: dG ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.21 M ; Experimental Assay: dG buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.57 M, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: dG ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.96 M ; Experimental Assay: dG ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.63 M ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.47 M, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.31 M, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.15 M, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: dG buffers:Tris, Gly: 25 mM, 192 mM, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: Tm units:°C, ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.83 M ; Experimental Assay: Tm units:°C, ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.21 M ; Experimental Assay: Tm buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.57 M, units:°C, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: Tm units:°C, ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.96 M ; Experimental Assay: Tm units:°C, ionic:NaCl,CaCl2: 25 mM,35 microM, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.63 M ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.47 M, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.31 M, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.15 M, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: Tm units:°C, ionic:NaCl,CaCl2: 25 mM,35 microM ; Experimental Assay: dG buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.52 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.21 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.89 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.57 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.29 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.80 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.63 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.47 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.31 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.15 M, ionic:NaCl: 30 mM ; Experimental Assay: dG buffers:Tris, Gly: 25 mM, 192 mM, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.52 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 1.21 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.89 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.57 M, units:°C, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, fructose: 25 mM, 192 mM, 0.29 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.80 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.63 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.47 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.31 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, buffers:Tris, Gly, sucrose: 25 mM, 192 mM, 0.15 M, ionic:NaCl: 30 mM ; Experimental Assay: Tm units:°C, ionic:NaCl: 30 mM
Libraries Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL ; Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV

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
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
1F6R 2000-06-23T00:00:00+0000 2.2 CRYSTAL STRUCTURE OF APO-BOVINE ALPHA-LACTALBUMIN
1F6S 2000-06-23T00:00:00+0000 2.2 CRYSTAL STRUCTURE OF BOVINE ALPHA-LACTALBUMIN
1FKQ 2000-08-10T00:00:00+0000 1.8 RECOMBINANT GOAT ALPHA-LACTALBUMIN T29V
1FKV 2000-08-10T00:00:00+0000 2.0 RECOMBINANT GOAT ALPHA-LACTALBUMIN T29I
1HFY 1996-06-13T00:00:00+0000 2.3 ALPHA-LACTALBUMIN
1HFZ 1996-06-13T00:00:00+0000 2.3 ALPHA-LACTALBUMIN
1HMK 1998-11-26T00:00:00+0000 2.0 RECOMBINANT GOAT ALPHA-LACTALBUMIN
2G4N 2006-02-22T00:00:00+0000 2.3 Anomalous substructure of alpha-lactalbumin
3B0K 2011-06-10T00:00:00+0000 1.6 Crystal structure of alpha-lactalbumin

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE
100.0 Alpha-lactalbumin P00711 LALBA_BOVIN
99.3 Alpha-lactalbumin Q9TSR4 LALBA_BOSMU
98.6 Alpha-lactalbumin Q9TSN6 LALBA_BUBBU
97.2 Alpha-lactalbumin P09462 LALBA_SHEEP
95.1 Alpha-lactalbumin P00712 LALBA_CAPHI