Graphene oxide as a protein matrix: influence on protein biophysical properties.
BACKGROUND
This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO.


RESULTS
Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation.


CONCLUSIONS
It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, 00931, USA. griselle.h.c@gmail.com.
Griselle
G
Hernández-Cancel
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, 00931, USA. suazo.damaris@gmail.com.
Dámaris
D
Suazo-Dávila
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, 00931, USA. ajo_1122@yahoo.com.
Axel J
AJ
Ojeda-Cruzado
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, 00931, USA. desiregt@gmail.com.
Desiree
D
García-Torres
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, 00931, USA. carlos.cabrera2@upr.edu.
Carlos R
CR
Cabrera
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, 00931, USA. kai.griebenow@gmail.com.
Kai
K
Griebenow
D055162:Biocatalysis; D055592:Biophysical Phenomena; D002208:Caproates / Q000737:chemistry; D002942:Circular Dichroism; D041961:Deuterium Exchange Measurement; D003911:Dextrans / Q000737:chemistry; D006031:Glycosylation; D006108:Graphite / Q000737:chemistry*; D055767:Immobilized Proteins / Q000378:metabolism; D007700:Kinetics; D053758:Nanoparticles / Q000737:chemistry; D010084:Oxidation-Reduction; D010087:Oxides / Q000737:chemistry*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry*; D056951:Photoelectron Spectroscopy; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D013696:Temperature
D002208:Caproates; D003911:Dextrans; D055767:Immobilized Proteins; D010087:Oxides; C037652:hexanoic acid; D006108:Graphite; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase