Graphene oxide as a protein matrix: influence on protein biophysical properties

Journal of Nanobiotechnology

Kai Griebenow¹  Carlos R. Cabrera¹  Desiree García-Torres¹  Axel J. Ojeda-Cruzado¹  Dámaris Suazo-Dávila¹  Griselle Hernández-Cancel¹ 

[1] Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan 00931, PR, USA

关键词: Thermostability;    Structural protein dynamic;    Glycosylation;    Graphene oxide;    Bilirubin oxidase;   

Others  :  1231706
DOI  :  10.1186/s12951-015-0134-0

 received in 2015-08-10, accepted in 2015-10-08,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Hernández-Cancel et al.

【 图 表 】

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【 参考文献 】

• [1]Pattammattel A, Puglia M, Chakraborty S, Deshapriya IK, Dutta PK, Kumar CV. Tuning the activities and structures of enzymes bound to graphene oxide with a protein glue. Langmuir. 2013; 29:15643-15654.
• [2]Zhang Y, Zhang J, Huang X, Zhou X, Wu H, Guo S. Assembly of graphene oxide-enzyme conjugates through hydrophobic interaction. Small. 2012; 8:154-159.
• [3]Zhang J, Zhang F, Yang H, Huang X, Liu H, Zhang J, Guo S. Graphene oxide as a matrix for enzyme immobilization. Langmuir. 2010; 26:6083-6085.
• [4]Mittag T, Kay LE, Forman-Kay JD. Protein dynamics and conformational disorder in molecular recognition. J Mol Recognit. 2010; 23:105-116.
• [5]Kempner ES. Movable lobes and flexible loops in proteins structural deformations that control biochemical activity. FEBS Lett. 1993; 326:4-10.
• [6]Sola RJ, Griebenow K. Influence of modulated structural dynamics on the kinetics of alpha-chymotrypsin catalysis. Insights through chemical glycosylation, molecular dynamics and domain motion analysis. FEBS J. 2006; 273:5303-5319.
• [7]Sola RJ, Griebenow K. Chemical glycosylation: new insights on the interrelation between protein structural mobility, thermodynamic stability, and catalysis. FEBS Lett. 2006; 580:1685-1690.
• [8]Pagan M, Sola RJ, Griebenow K. On the role of protein structural dynamics in the catalytic activity and thermostability of serine protease subtilisin Carlsberg. Biotechnol Bioeng. 2009; 103:77-84.
• [9]Huang J, Gao X, Jia J, Kim JK, Li Z. Graphene oxide-based amplified fluorescent biosensor for Hg(2+) detection through hybridization chain reactions. Anal Chem. 2014; 86:3209-3215.
• [10]Ambrosi A, Sasaki T, Pumera M. Platelet graphite nanofibers for electrochemical sensing and biosensing: the influence of graphene sheet orientation. Chem Asian J. 2010; 5:266-271.
• [11]Das D, Ghosh S, Basumallick I. Electrochemical studies on glucose oxidation in an enzymatic fuel cell with enzyme immobilized on to reduced graphene Oxide surface. Electroanalysis. 2014; 26:2408-2418.
• [12]Sun X, Liu Z, Welsher K, Robinson JT, Goodwin A, Zaric S, Dai H. Nano-graphene oxide for cellular imaging and drug delivery. Nano Res. 2008; 1:203-212.
• [13]Liu J, Cui L, Losic D. Graphene and graphene oxide as new nanocarriers for drug delivery applications. Acta Biomater. 2013; 9:9243-9257.
• [14]Bao H, Pan Y, Ping Y, Sahoo NG, Wu T, Li L, Li J, Gan LH. Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery. Small. 2011; 7:1569-1578.
• [15]Shao Y, Wang J, Wu H, Liu J, Aksay IA, Lin Y. Graphene based electrochemical sensors and biosensors: a review. Electroanalysis. 2010; 22:1027-1036.
• [16]Shen H, Zhang L, Liu M, Zhang Z. Biomedical applications of graphene. Theranostics. 2012; 2:283-294.
• [17]Wang Y, Li Z, Wang J, Li J, Lin Y. Graphene and graphene oxide: biofunctionalization and applications in biotechnology. Trends Biotechnol. 2011; 29:205-212.
• [18]Jin L, Yang K, Yao K, Zhang S, Tao H, Lee ST, Liu Z, Peng R. Functionalized graphene oxide in enzyme engineering: a selective modulator for enzyme activity and thermostability. ACS Nano. 2012; 6:4864-4875.
• [19]Alava T, Mann JA, Theodore C, Benitez JJ, Dichtel WR, Parpia JM, Craighead HG. Control of the graphene-protein interface is required to preserve adsorbed protein function. Anal Chem. 2013; 85:2754-2759.
• [20]Zhou L, Jiang Y, Gao J, Zhao X, Ma L, Zhou Q. Oriented immobilization of glucose oxidase on graphene oxide. Biochem Eng J. 2012; 69:28-31.
• [21]Delgado Y, Morales-Cruz M, Hernandez-Roman J, Martinez Y, Griebenow K. Chemical glycosylation of cytochrome c improves physical and chemical protein stability. BMC Biochem. 2014; 15:16. BioMed Central Full Text
• [22]Hernandez-Cancel G, Suazo-Davila D, Medina-Guzman J, Rosado-Gonzalez M, Diaz-Vazquez LM, Griebenow K. Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor. Anal Chim Acta. 2015; 854:129-139.
• [23]Mendez J, Morales Cruz M, Delgado Y, Figueroa CM, Orellano EA, Morales M, Monteagudo A, Griebenow K. Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells. Mol Pharm. 2014; 11:102-111.
• [24]Flores-Fernandez GM, Griebenow K. Glycosylation improves alpha-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic)acid microspheres. Results Pharma Sci. 2012; 2:46-51.
• [25]Huang J, Guerrero A, Parker E, Strum JS, Smilowitz JT, German JB, Lebrilla CB. Site-specific glycosylation of secretory immunoglobulin A from human colostrum. J Proteome Res. 2015; 14:1335-1349.
• [26]Wormald MR, Rudd PM, Harvey DJ, Chang SC, Scragg IG, Dwek RA. Variations in oligosaccharide-protein interactions in immunoglobulin G determine the site-specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides. Biochemistry. 1997; 36:1370-1380.
• [27]Shao Q, Wu P, Xu X, Zhang H, Cai C. Insight into the effects of graphene oxide sheets on the conformation and activity of glucose oxidase: towards developing a nanomaterial-based protein conformation assay. Phys Chem Chem Phys. 2012; 14:9076-9085.
• [28]Yao YD, Sun TM, Huang SY, Dou S, Lin L, Chen JN, Ruan JB, Mao CQ, Yu FY, Zeng MS, Zang JY et al.. Targeted delivery of PLK1-siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis. Sci Transl Med. 2012; 4:130ra48.
• [29]Batra B, Lata S, Sunny, Rana JS, Pundir CS. Construction of an amperometric bilirubin biosensor based on covalent immobilization of bilirubin oxidase onto zirconia coated silica nanoparticles/chitosan hybrid film. Biosens Bioelectron. 2013; 44:64-69.
• [30]Kannan P, Chen H, Lee VT, Kim DH. Highly sensitive amperometric detection of bilirubin using enzyme and gold nanoparticles on sol-gel film modified electrode. Talanta. 2011; 86:400-407.
• [31]Omar Garcia S, Narváez Villarrubia C, Falase A, Atanassov P. Gas-diffusion cathodes integrating carbon nanotube modified-Toray paper and bilirubin oxidase. J Electrochem Soc. 2014; 161:H523-H528.
• [32]Kim J, Parkey J, Rhodes C, Gonzalez-Martin A. Development of a biofuel cell using glucose-oxidase- and bilirubin-oxidase-based electrodes. J Solid State Electrochem. 2009; 13:1043-1050.
• [33]Dos Santos L, Climent V, Blanford CF, Armstrong FA. Mechanistic studies of the ‘blue’ Cu enzyme, bilirubin oxidase, as a highly efficient electrocatalyst for the oxygen reduction reaction. Phys Chem Chem Phys. 2010; 12:13962-13974.
• [34]Mano N. Features and applications of bilirubin oxidases. Appl Microbiol Biotechnol. 2012; 96:301-307.
• [35]Mano N, Edembe L. Bilirubin oxidases in bioelectrochemistry: features and recent findings. Biosens Bioelectron. 2013; 50:478-485.
• [36]Samejima T, Wu CS, Shiboya K, Kaji H, Koikeda S, Ando K, Yang JT. Conformation of bilirubin oxidase in native and denatured states. J Protein Chem. 1994; 13:307-313.
• [37]Habeeb AF. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal Biochem. 1966; 14:328-336.
• [38]Arakawa T, Prestrelski SJ, Kenney WC, Carpenter JF. Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev. 2001; 46:307-326.
• [39]Henzler-Wildman KA, Lei M, Thai V, Kerns SJ, Karplus M, Kern D. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature. 2007; 450:913-916.
• [40]Gotoh Y, Kondo Y, Kaji H, Takeda A, Samejima T. Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses. J Biochem. 1989; 106:621-626.
• [41]Jackson M, Mantsch HH. The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol. 1995; 30:95-120.
• [42]Ferraro DM, Lazo N, Robertson AD. EX1 hydrogen exchange and protein folding. Biochemistry. 2004; 43:587-594.
• [43]Castillo B, Sola RJ, Ferrer A, Barletta G, Griebenow K. Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4-dioxane. Biotechnol Bioeng. 2008; 99:9-17.
• [44]Shental-Bechor D, Levy Y. Communication: folding of glycosylated proteins under confinement. J Chem Phys. 2011; 135:141104.
• [45]England J, Lucent D, Pande V. Rattling the cage: computational models of chaperonin-mediated protein folding. Curr Opin Struct Biol. 2008; 18:163-169.
• [46]Klimov DK, Newfield D, Thirumalai D. Simulations of β-hairpin folding confined to spherical pores using distributed computing. Proc Natl Acad Sci. 2002; 99:8019-8024.
• [47]Sola RJ, Rodriguez-Martinez JA, Griebenow K. Modulation of protein biophysical properties by chemical glycosylation: biochemical insights and biomedical implications. Cell Mol Life Sci. 2007; 64:2133-2152.
• [48]Cunci L, Velez CA, Perez I, Suleiman A, Larios E, Jose-Yacaman M, Watkins JJ, Cabrera CR. Platinum electrodeposition at unsupported electrochemically reduced nanographene oxide for enhanced ammonia oxidation. ACS Appl Mater Interfaces. 2014; 6:2137-2145.
• [49]Rodriguez-Martinez JA, Sola RJ, Castillo B, Cintron-Colon HR, Rivera-Rivera I, Barletta G, Griebenow K. Stabilization of alpha-chymotrypsin upon PEGylation correlates with reduced structural dynamics. Biotechnol Bioeng. 2008; 101:1142-1149.
• [50]Zavodszky P, Johansen JT, Hvidt A. Hydrogen-exchange study of the conformational stability of human carbonic-anhydrase B and its metallocomplexes. Eur J Biochem. 1975; 56:67-72.