Understanding the Effect of Single Cysteine Mutations on Gold Nanoclusters as Studied by Spectroscopy and Density Functional Theory Modeling

Abstract

Fluorescent metal nanoclusters have generated considerable excitement in nanobiotechnology, particularly in the applications of biolabeling, targeted delivery, and biological sensing. The present work is an experimental and computational study that aims to understand the effects of protein environment on the synthesis and electronic properties of gold nanoclusters. MPT63, a drug target of Mycobacterium tuberculosis, was used as the template protein to synthesize, for the first time, gold nanoclusters at a low micromolar concentration of the protein. Two single cysteine mutants of MPT63, namely, MPT63Gly20Cys (mutant I) and MPT63Gly40Cys (mutant II) were employed for this study. The experimental results show that cysteine residues positioned in two different regions of the protein induce varying electronic states of the nanoclusters depending on the surrounding amino acids. A mixture of five-atom and eight-atom clusters was generated for each mutant, and the former was found to be predominant in both cases. Computational studies, including density functional theory (DFT), frontier molecular orbital (FMO), and natural bond orbital (NBO) calculations, validated the experimental observations. The as-prepared protein-stabilized nanoclusters were found to have applications in the imaging of live cells.

A New Phase Transfer Strategy to Convert Protein‐Capped Nanomaterials into Uniform Fluorescent Nanoclusters in Reverse Micellar Phase

“…Following the synthesis of bovine serum albumin (BSA) protected fluorescent gold nanocluster by Xie and co‐workers, many researches utilized the procedure to prepare MNCs with various combinations of proteins and metals ,. Use of external reducing agent incorporates further flexibility to generate multicolored fluorescent nanoclusters which are even inter‐convertible under proper conditions . Diverse applications and extreme popularity of this method demand more advancement, in particular, its expansion into the organic media.…”

A Simple Approach to Design Proteins for the Sustainable Synthesis of Metal Nanoclusters

“…[4] Metal NCs need to be stabilized by different molecules, such as dendrimers, [5] small molecules, [6] DNA, [7] peptides, [8] and proteins. [20] However, asimple approach to design artificial and/or natural proteins with an incorporated predefined metal binding site for the synthesis of metal NCs is still not available.T odate,there are only two examples reported that explored the rational introduction of amino acids for cluster coordination:t he insertion of as ingle cysteine (Cys) for the of metal NCs, [21] and the incorporation of two additional Cys residues in the ferritin cage to enhance the uptake of Au ions. [8,10] Thiol, amine,a nd carboxylic acid side-chain functionalities seem to promote metal binding.…”

“…[9] NCs stabilized by peptides and proteins can be produced under mild conditions,a nd the obtained structures are stable under aw ide range of pH values and ionic forces, making them ideal for biological applications.N atural and designed histidine-rich and cysteine-rich peptides have been used for the synthesis and stabilization of metal NCs. [20] However, asimple approach to design artificial and/or natural proteins with an incorporated predefined metal binding site for the synthesis of metal NCs is still not available.T odate,there are only two examples reported that explored the rational introduction of amino acids for cluster coordination:t he insertion of as ingle cysteine (Cys) for the of metal NCs, [21] and the incorporation of two additional Cys residues in the ferritin cage to enhance the uptake of Au ions. [11] Different commercially available proteins,s uch as bovine serum albumin (BSA), [12] papain, [13] human transferrin, [14] lysozyme, [15] trypsin, [16] pepsin, [17] insulin, [18] and peroxidase, [19] have been employed in the preparation of metal NCs.M ajor efforts have been made to understand the role of protein characteristics, including protein size and amino acid content, in the formation and properties of metal NCs;t he metal coordination environments that promote biomineralization;a nd the NCs structure and formation mechanisms as well as interactions of the metal core with the protein.…”

“…[8,10] Thiol, amine,a nd carboxylic acid side-chain functionalities seem to promote metal binding. [20] However, asimple approach to design artificial and/or natural proteins with an incorporated predefined metal binding site for the synthesis of metal NCs is still not available.T odate,there are only two examples reported that explored the rational introduction of amino acids for cluster coordination:t he insertion of as ingle cysteine (Cys) for the of metal NCs, [21] and the incorporation of two additional Cys residues in the ferritin cage to enhance the uptake of Au ions. [20] However, asimple approach to design artificial and/or natural proteins with an incorporated predefined metal binding site for the synthesis of metal NCs is still not available.T odate,there are only two examples reported that explored the rational introduction of amino acids for cluster coordination:t he insertion of as ingle cysteine (Cys) for the of metal NCs, [21] and the incorporation of two additional Cys residues in the ferritin cage to enhance the uptake of Au ions.…”

“…[11] Different commercially available proteins,s uch as bovine serum albumin (BSA), [12] papain, [13] human transferrin, [14] lysozyme, [15] trypsin, [16] pepsin, [17] insulin, [18] and peroxidase, [19] have been employed in the preparation of metal NCs.M ajor efforts have been made to understand the role of protein characteristics, including protein size and amino acid content, in the formation and properties of metal NCs;t he metal coordination environments that promote biomineralization;a nd the NCs structure and formation mechanisms as well as interactions of the metal core with the protein. [20] However, asimple approach to design artificial and/or natural proteins with an incorporated predefined metal binding site for the synthesis of metal NCs is still not available.T odate,there are only two examples reported that explored the rational introduction of amino acids for cluster coordination:t he insertion of as ingle cysteine (Cys) for the of metal NCs, [21] and the incorporation of two additional Cys residues in the ferritin cage to enhance the uptake of Au ions. [20c] Thec urrent study explores the design of ac luster coordination site into ap rotein structure as as imple and versatile approach for the sustainable synthesis and stabilization of metal NCs into selected proteins.I np articular,w e used repeat proteins as an artificial protein system but we believe that this method can be used as ageneral approach to incorporate cluster coordination sites into functional proteins to expand their applications.T he resulting designed repeat proteins were evaluated as templates for the synthesis and stabilization of NCs with different metal composition ( Figure 1).…”