Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.


Abstract

We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions. Study holds ProTherm entries: 15120, 15121, 15122, 15123, 15124, 15125, 15126, 15127, 15128, 15129, 15130, 15131, 15132, 15133, 15134 Extra Details: sucrose; ligands binding; thermodynamic stability; hydrogen-deuterium exchange

Submission Details

ID: N6fSEiXS3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Krishnan S;Chi EY;Webb JN;Chang BS;Shan D;Goldenberg M;Manning MC;Randolph TW;Carpenter JF,Biochemistry (2002) Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition. PMID:12009905
Additional Information

Number of data points 20
Proteins Granulocyte colony-stimulating factor ; Granulocyte-macrophage colony-stimulating factor
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dG details:Additives sucrose (1.0 M), ; Experimental Assay: dG details:Additives sucrose (0.75 M), ; Experimental Assay: dG details:Additives sucrose (0.5 M), ; Experimental Assay: dG details:Additives sucrose (0.25 M), ; Experimental Assay: dG details:Additives ; Experimental Assay: Tm details:Additives sucrose (1.0 M), ; Experimental Assay: dHvH details:Additives sucrose (1.0 M), ; Experimental Assay: Tm details:Additives sucrose (0.75 M), ; Experimental Assay: dHvH details:Additives sucrose (0.75 M), ; Experimental Assay: Tm details:Additives sucrose (0.5 M), ; Experimental Assay: dHvH details:Additives sucrose (0.5 M), ; Experimental Assay: Tm details:Additives sucrose (0.25 M), ; Experimental Assay: dHvH details:Additives sucrose (0.25 M), ; Experimental Assay: Tm details:Additives ; Experimental Assay: dHvH details:Additives ; Experimental Assay: Cm details:Additives sucrose (1.0M), ; Experimental Assay: Cm details:Additives sucrose (0.75M), ; Experimental Assay: Cm details:Additives sucrose (0.5M), ; Experimental Assay: Cm details:Additives sucrose (0.25M), ; Experimental Assay: Cm details:Additives
Libraries Mutations for sequence TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP
Sequence Assay Result Units