Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony‐stimulating factor

We studied the non‐native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (ΔGunf) and osmotic second virial coefficient (B22), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ΔGunf and negative B22), the first step is rate‐limiting, and increasing ΔGunf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B22 values) the second step is rate‐limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate‐limiting.

[1]  C. Zukoski,et al.  Protein interactions and phase behavior: Sensitivity to the form of the pair potential , 1999 .

[2]  J. Carpenter,et al.  Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  N. Elvassore,et al.  Forces between aqueous nonuniformly charged colloids from molecular simulation , 2002 .

[4]  Zamora,et al.  Phase behavior of small attractive colloidal particles. , 1996, Physical review letters.

[5]  A. V. Smith,et al.  Protein refolding versus aggregation: computer simulations on an intermediate-resolution protein model. , 2001, Journal of molecular biology.

[6]  W. William Wilson,et al.  Correlation of second virial coefficients and solubilities useful in protein crystal growth , 1999 .

[7]  A. Lenhoff,et al.  Measured and calculated effects of mutations in bacteriophage T4 lysozyme on interactions in solution , 2000, Proteins.

[8]  Kelly K. Lee,et al.  Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein , 2002, Protein science : a publication of the Protein Society.

[9]  Abraham M Lenhoff,et al.  Self-interaction chromatography: a novel screening method for rational protein crystallization. , 2002, Acta crystallographica. Section D, Biological crystallography.

[10]  B. Chang,et al.  Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: role in restricted conformational mobility and compaction of native state. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  W. William Wilson,et al.  Relation between the solubility of proteins in aqueous solutions and the second virial coefficient of the solution , 1999 .

[12]  R. Sousa.  Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization. , 1995, Acta crystallographica. Section D, Biological crystallography.

[13]  A. Arabshahi,et al.  [6] Second virial coefficient as predictor in protein crystal growth. , 1997, Methods in enzymology.

[14]  J. Carpenter,et al.  A transient expansion of the native state precedes aggregation of recombinant human interferon-gamma. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D Eisenberg,et al.  The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Manning,et al.  Counteracting Effects of Renal Solutes on Amyloid Fibril Formation by Immunoglobulin Light Chains* , 2001, The Journal of Biological Chemistry.

[17]  J. Philo,et al.  Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states. , 2001, Analytical biochemistry.

[18]  A George,et al.  Predicting protein crystallization from a dilute solution property. , 1994, Acta crystallographica. Section D, Biological crystallography.

[19]  T. Arakawa,et al.  Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride , 1991, Journal of protein chemistry.

[20]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[21]  G Shimamoto,et al.  Chemical modification and site-directed mutagenesis of methionine residues in recombinant human granulocyte colony-stimulating factor: effect on stability and biological activity. , 1999, Archives of biochemistry and biophysics.

[22]  Y. Shamoo,et al.  Photochemical crosslinking of bacteriophage T4 single‐stranded DNA‐binding protein (gp32) to oligo‐p(dT)8: Identification of phenylalanine‐183 as the site of crosslinking , 1988, Proteins.

[23]  P. C. Hiemenz,et al.  Principles of colloid and surface chemistry , 1977 .

[24]  O. Velev,et al.  Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen. , 1998, Biophysical journal.

[25]  D. A. Harris,et al.  Principles of Biochemistry (2nd edn) , 1993 .

[26]  C. Pace.  Measuring and increasing protein stability. , 1990, Trends in biotechnology.

[27]  A. Lenhoff,et al.  Molecular origins of osmotic second virial coefficients of proteins. , 1998, Biophysical journal.

[28]  Michael Farnum,et al.  Effect of glycerol on the interactions and solubility of bovine pancreatic trypsin inhibitor. , 1999, Biophysical journal.

[29]  Theodore W Randolph,et al.  Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition. , 2002, Biochemistry.

[30]  N. Denkov,et al.  Diffusion of charged colloidal particles at low volume fraction: Theoretical model and light scattering experiments , 1992 .

[31]  R. Borchardt,et al.  Stability of Protein Pharmaceuticals , 1989, Pharmaceutical Research.

[32]  S. Yau,et al.  Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes. , 2000, Biophysical journal.

[33]  Pace Cn,et al.  Measuring and increasing protein stability , 1990 .

[34]  A. Fink.  Protein aggregation: folding aggregates, inclusion bodies and amyloid. , 1998, Folding & design.

[35]  Kevin L. Shaw,et al.  Linear extrapolation method of analyzing solvent denaturation curves , 2000, Proteins.

[36]  E. M.,et al.  Statistical Mechanics , 2021, Manual for Theoretical Chemistry.

[37]  Jan Drenth,et al.  Understanding protein crystallization on the basis of the phase diagram , 1999 .

[38]  Håkan Wennerström,et al.  Role of hydration and water structure in biological and colloidal interactions , 1996, Nature.

[39]  A. Gittis,et al.  Protein crystallization by design: chymotrypsinogen without precipitants. , 2000, Journal of molecular biology.

[40]  M. Manning,et al.  Comparative fourier transform infrared and circular dichroism spectroscopic analysis of alpha1-proteinase inhibitor and ovalbumin in aqueous solution. , 2000, Archives of biochemistry and biophysics.