title: Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles.
abstract: Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis-isolated as an endophytic fungus from Rizophora mangle-were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon their physical interaction with the AgNPs or when covalently bonded to them. Eight proteins in the AgNP dispersion were identified by mass spectrometry analyses. All these proteins are involved in metabolic pathways of the fungus and are important for carbon, phosphorous and nitrogen uptake, and for the fungal growth. Thereby, important proteins for fungi are also involved in the formation and stabilization of the biogenic AgNPs.
authors.affiliation: Laboratório de Química Biológica, Instituto de Química, Universidade Estadual de Campinas, Campinas, SP, Brazil.|NanoBioss, SisNano, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: Daniela
authors.initials: D
authors.lastname: Ballottin
authors.affiliation: Laboratório de Química Biológica, Instituto de Química, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Fulaz
authors.affiliation: Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.|Instituto de Ciências Exatas, Universidade Federal Fluminense, Volta Redonda, RJ, Brazil.
authors.forename: Michele L
authors.initials: ML
authors.lastname: Souza
authors.affiliation: Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Paola
authors.initials: P
authors.lastname: Corio
authors.affiliation: Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Alexandre G
authors.initials: AG
authors.lastname: Rodrigues
authors.affiliation: Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Ana O
authors.initials: AO
authors.lastname: Souza
authors.affiliation: Laboratório de Nanobiotecnologia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Riberão Preto, SP, Brazil.
authors.forename: Priscyla M
authors.initials: PM
authors.lastname: Gaspari
authors.affiliation: Laboratório Dalton, Instituto de Química, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: Alexandre F
authors.initials: AF
authors.lastname: Gomes
authors.affiliation: Laboratório Dalton, Instituto de Química, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: Fábio
authors.initials: F
authors.lastname: Gozzo
authors.affiliation: Laboratório de Química Biológica, Instituto de Química, Universidade Estadual de Campinas, Campinas, SP, Brazil. ljubica@iqm.unicamp.br.|NanoBioss, SisNano, Universidade Estadual de Campinas, Campinas, SP, Brazil. ljubica@iqm.unicamp.br.
authors.forename: Ljubica
authors.initials: L
authors.lastname: Tasic
keywords: Aspergillus tubingensis; Biogenic silver nanoparticles (AgNPs); Capping proteins