Premium
Stabilization of α‐chymotrypsin upon PEGylation correlates with reduced structural dynamics
Author(s) -
RodríguezMartínez José A.,
Solá Ricardo J.,
Castillo Betzaida,
CintrónColón Héctor R.,
RiveraRivera Izarys,
Barletta Gabriel,
Griebenow Kai
Publication year - 2008
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22014
Subject(s) - pegylation , chemistry , molecular dynamics , peg ratio , chymotrypsin , protein structure , biophysics , structural stability , covalent bond , circular dichroism , trypsin , protein tertiary structure , polyethylene glycol , crystallography , computational chemistry , enzyme , biochemistry , organic chemistry , biology , economics , structural engineering , engineering , finance
Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing. To test this hypothesis, the model protein α‐chymotrypsin (α‐CT) was covalently modified with PEGs with molecular weights ( M W ) of 700, 2,000 and 5,000 and the degree of modification was systematically varied. The procedure did not cause significant tertiary structure changes. Thermodynamic unfolding experiments revealed that PEGylation increased the thermal transition temperature ( T m ) of α‐CT by up to 6°C and the free energy of unfolding [Δ G U (25°C)] by up to 5 kcal/mol. The increase in stability was found to be independent of the PEG M W and it leveled off after an average of four PEG molecules were bound to α‐CT. Fourier‐transformed infrared (FTIR) H/D exchange experiments were conducted to characterize the conformational dynamics of the PEG‐conjugates. It was found that the magnitude of thermodynamic stabilization correlates with a reduction in protein structural dynamics and was independent of the PEG M W . Thus, the initial hypothesis proved positive. Similar to the thermodynamic stabilization of proteins by covalent modification with glycans, PEG thermodynamically stabilizes α‐CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc.