Erythropoietin unfolding: thermodynamics and its correlation with structural features.

Abstract

Human erythropoietin (EPO) is a glycoprotein hormone considered to be the principal regulator of red blood cell formation. Although its recombinant version (rEPO) has been widely used for treatment of various anemias and its biological effects are relatively well-known, we know little about its biophysical properties and their relation to its structure. To gain a fuller understanding of the structural and functional properties of rEPO on the molecular level we followed its thermal and urea-induced unfolding at different pH (3.1-9.4) and urea concentrations (0-8 M) using spectropolarimetry, UV absorption, intrinsic emission fluorescence, and differential scanning calorimetry. Our results show that under a variety of conditions rEPO undergoes thermal or urea-induced denaturation that may be considered as a reversible two-state process characterized by unusually high (thermal) or moderate (urea-induced) extent of the residual structure. The highest thermal stability of the protein observed in aqueous solutions at physiological pH appears to be due to the largest difference in the extent of structure in the denatured and native state at this pH. The comparison between experimentally determined energetics of rEPO denaturation and its structure-based calculations indicates that the parametrization of thermodynamic quantities in terms of changes in solvent accessible nonpolar and polar surface areas resulting from protein unfolding can be successfully used provided that these changes are estimated from combination of experimentally determined deltaC(o)p and deltaH(o) values and not calculated from the structure of the protein's folded and assumingly fully unfolded state. Study holds ProTherm entries: 19566, 19567, 19568, 19569, 19570, 19571, 19572, 19573, 19574, 19575, 19576, 19577, 19578, 19579, 19580, 19581, 19582, 19583 Extra Details: residual structure; solvent accessible; polar surface areas; unfolded state

Submission Details

ID: ZmRbH2n74

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Lah J;Prislan I;Krzan B;Salobir M;Francky A;Vesnaver G,Biochemistry (2005) Erythropoietin unfolding: thermodynamics and its correlation with structural features. PMID:16229477
Additional Information

Study Summary

Number of data points 48
Proteins Erythropoietin ; Erythropoietin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Tm pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: dHvH pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: Tm buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: dHvH buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: Tm pH:7.2, buffers:Phosphate: 50 mM ; Experimental Assay: dHvH pH:7.2, buffers:Phosphate: 50 mM ; Experimental Assay: Tm pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: dHvH pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: Tm pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: dHvH pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: Tm pH:3.1, buffers:Citrate: 50 mM ; Experimental Assay: dHvH pH:3.1, buffers:Citrate: 50 mM ; Experimental Assay: Tm pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: dHvH pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: Tm buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: dHvH buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: Tm pH:7.2, buffers:Phosphate: 50 mM ; Experimental Assay: dHvH pH:7.2, buffers:Phosphate: 50 mM ; Experimental Assay: Tm pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: dHvH pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: Tm pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: dHvH pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: Tm pH:3.1, buffers:Citrate: 50 mM ; Experimental Assay: dHvH pH:3.1, buffers:Citrate: 50 mM ; Experimental Assay: dCp pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: dHcal pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: Tm pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: dHvH pH:9.4, buffers:Borate: 20 mM ; Experimental Assay: dCp buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: dHcal buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: Tm buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: dHvH buffers:TRIS: 50 mM, pH:8.3 ; Experimental Assay: dCp pH:7.2, buffers:Phosphate: 50 mM ; Experimental Assay: dHcal pH:7.2, buffers:Phosphate: 50 mM ; Experimental Assay: Tm buffers:Phosphate: 50 mM, pH:7.2 ; Experimental Assay: dHvH buffers:Phosphate: 50 mM, pH:7.2 ; Experimental Assay: dCp pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: dHcal pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: Tm pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: dHvH pH:6.1, buffers:Cacodylate: 50 mM ; Experimental Assay: dCp pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: dHcal pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: Tm pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: dHvH pH:4.6, buffers:Acetate: 50 mM ; Experimental Assay: dCp pH:3.1, buffers:Citrate: 50 mM ; Experimental Assay: dHcal pH:3.1, buffers:Citrate: 50 mM ; Experimental Assay: Tm pH:3.1, buffers:Citrate: 50 mM ; Experimental Assay: dHvH pH:3.1, buffers:Citrate: 50 mM
Libraries Mutations for sequence APPRLICDSRVLERYLLEAKEAEKITTGCAEHCSLNEKITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVKSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

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 UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Erythropoietin P01588 EPO_HUMAN
91.7 Erythropoietin P07865 EPO_MACFA
90.7 Erythropoietin Q28513 EPO_MACMU