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

Study Summary

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

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
1GNC 1994-07-31 STRUCTURE AND DYNAMICS OF THE HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR DETERMINED BY NMR SPECTROSCOPY. LOOP MOBILITY IN A FOUR-HELIX-BUNDLE PROTEIN
5GW9 2017-09-13 1.65 Crystal structure of C163, a backbone circularized G-CSF
5ZO6 2019-04-17 1.7 Crystal structure of C166, a backbone circularized G-CSF
6BFS 2018-09-12 2.0 The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies
5D70 2015-10-14 2.06 Crystal structure of MOR03929, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
1RHG 1994-01-31 2.2 THE STRUCTURE OF GRANULOCYTE-COLONY-STIMULATING FACTOR AND ITS RELATIONSHIP TO THOSE OF OTHER GROWTH FACTORS
5D71 2015-10-14 2.25 Crystal structure of MOR04302, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
2GMF 1996-11-08 2.4 HUMAN GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR
5D72 2015-10-14 2.6 Crystal structure of MOR04252, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
6BFQ 2018-09-12 2.6 The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies
4RS1 2015-11-11 2.68 Crystal structure of receptor-cytokine complex
1CSG 1993-10-31 2.7 Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor
2D9Q 2006-02-07 2.8 Crystal Structure of the Human GCSF-Receptor Signaling Complex
1CD9 2000-03-08 2.8 2:2 COMPLEX OF G-CSF WITH ITS RECEPTOR
5C7X 2015-10-14 2.95 Crystal structure of MOR04357, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
4NKQ 2015-09-23 3.3 Structure of a Cytokine Receptor Complex
1PGR 2000-03-08 3.5 2:2 COMPLEX OF G-CSF WITH ITS RECEPTOR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
98.3 Granulocyte colony-stimulating factor P09919 CSF3_HUMAN
94.4 Granulocyte-macrophage colony-stimulating factor Q0MUT8 CSF2_CHLAE
100.0 Granulocyte-macrophage colony-stimulating factor P04141 CSF2_HUMAN