Stabilization of alpha-chymotrypsin upon PEGylation correlates with reduced structural dynamics.
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 alpha-chymotrypsin (alpha-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 alpha-CT by up to 6 degrees C and the free energy of unfolding [DeltaG(U) (25 degrees 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 alpha-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 alpha-CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations.
Rodríguez-Martínez
José A
JA
Department of Chemistry, University of Puerto Rico-Río Piedras, San Juan, Puerto Rico.
Solá
Ricardo J
RJ
Castillo
Betzaida
B
Cintrón-Colón
Héctor R
HR
Rivera-Rivera
Izarys
I
Barletta
Gabriel
G
Griebenow
Kai
K
D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D005583:Fourier Analysis; D011092:Polyethylene Glycols / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013696:Temperature
D011092:Polyethylene Glycols; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
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