Engineering and design in the bioelectrochemistry of metalloproteins

doi:10.1016/S0959-440X(00)00238-4

Current Opinion in Structural Biology

Volume 11, Issue 4, 1 August 2001, Pages 491-499

https://doi.org/10.1016/S0959-440X(00)00238-4 Get rights and content

Abstract

Engineered metalloproteins offer interesting systems for electrochemical studies of protein structure/function and their applications in nanobiotechnology. Scanning probe microscopy and cyclic voltammetry of engineered metalloproteins and electrodes have proved to be a powerful combination of tools contributing to the field of bioelectrochemistry. The ability to engineer tags, such as histidine tags and biotin-acceptor peptides, and to site-specifically introduce cysteine residues enabled the creation of ordered immobilised protein structures that can be characterised both electrochemically and topographically. Gene fusion and de novo combinatorial synthesis of metalloproteins are emerging to provide structures with the desired electrochemical properties.

Introduction

Over the past 20 years, electrochemistry has proven to be both a useful means to understand the biochemistry of metalloproteins and enzymes, and a powerful method for the exploitation of these proteins in biosensors and bioelectronics. The principles underpinning electrochemical methods are well established and knowledge of the structure/function of metalloproteins is increasingly available: these two factors allow the merging of two areas of research that are traditionally domains of chemists and biologists into the innovative discipline of bioelectrochemistry. Recent developments of this field are documented in the proceedings of the General Discussion on Bioelectrochemistry of the Royal Society of Chemistry, held at the University of Southampton in July 2000 [1].

An exciting recent development in this area is the application of protein design and engineering to tailor new electrochemical devices and to better understand protein–electrode interaction [2••]. Over the past three years, bioelectrochemistry has provided valuable contributions to better understand the structure/function of redox proteins, as well as to characterise new redox functions engineered either by the modification of existing proteins or by de novo construction from first principles. Another strong area of research is the tailoring of electrode surfaces to produce controlled and sensitive biolayers by engineering the surface of either the proteins or the electrode, or a combination of both, with biocompatible elements (Fig. 1).

This article will review some of the developments over the past three years in these areas of research.

Section snippets

Electrochemistry of designed metalloproteins

Electrochemical and spectroelectrochemical methods can assist in understanding the structure/function relationships of mutated, as well as de novo designed, metalloproteins and enzymes. The design of metalloproteins from first principles is a fascinating area of research that has also provided interesting systems to study the determinants of redox properties, as well as new structural elements that are able to host metal centres. An example of these systems is given by the four-helix bundles

Electrochemical characterisation ofmetalloproteins

Here we will briefly review some of the strategies developed to address electrochemically challenging metalloproteins, including the innovative application of protein film voltammetry (PFV).

Direct electrochemistry was developed in the late 1970s as a means to study redox proteins using electrochemical techniques. Early experiments with cytochrome c were carried out using gold or various graphite electrodes. During the past two decades, direct electrochemistry has developed into the method of

Engineering metalloproteins for immobilisation

The controlled and oriented immobilisation of metalloproteins on electrode surfaces is an attractive goal in bioelectrochemical research. The main objective is to achieve rapid and efficient interfacial electron transfer rates (k_ET) between the metalloprotein and the electrode surface. This is undoubtedly important for the construction of efficient nanodevices, such as fuel cells, artificial photosynthetic systems, bioelectronics and biosensors of rapidly degrading analytes such as oxygen

Engineering electrode surfaces for immobilisation of metalloproteins

Nonspecific direct immobilisation of proteins onto a metal substrate presents several disadvantages, such as denaturation, unstable binding leading to exchange events and reversibility of binding, and unspecific, random and multioriented immobilisation. The chemical modification of the surface of either the electrode or the protein may increase the stability of the protein and introduce the possibility of controlling the density and environment of the immobilised species. It has been shown that

Conclusions

Progress made in the areas of enzyme-modified electrodes [48], microelectrode array devices [49] and the possibility of rapid automated screening of electrode arrays [50] is converging to provide exciting developments in bioelectrochemistry. Protein engineering has started to provide a valuable contribution towards the design of the surface and the functional properties of metalloproteins. This, combined with the controlled chemical modification of electrodes, is able to achieve a designed

Acknowledgements

The Human Science Frontiers Programme (grant RG-44/98) and the BBSRC (grant 28/E09651) are gratefully acknowledged for financial support.

References and recommended reading

Papers of particular interest, published within the annual period of review,have been highlighted as:

•of special interest
••of outstanding interest

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ReviewEngineering and design in the bioelectrochemistry of metalloproteins

Abstract

Introduction

Section snippets

Electrochemistry of designed metalloproteins

Electrochemical characterisation ofmetalloproteins

Engineering metalloproteins for immobilisation

Engineering electrode surfaces for immobilisation of metalloproteins

Conclusions

Acknowledgements

References and recommended reading

Coord Chem Rev

JElectroanal Chem

Bioelectrochem Bioenerg

Electrochem Commun

Electrochim Acta

J Electroanal Chem

Anal Chim Acta

Biosens Bioelectron

FEBS Lett

Electrochem Commun

Trac-Trends Anal Chem

Anal Biochem

Biosens Bioelectron

Biosens Bioelectron

Electrochim Acta

J Electroanal Chem

Electrochim Acta

Biosens Bioelectron

Biosens Bioelectron

J Electroanal Chem

Combinatorial synthesis of four-helix bundle hemoproteins for tuning of cofactor properties

Angew Chem Int Ed Engl

De novo design and characterization of copper centers in synthetic four-helix-bundle proteins

J Am Chem Soc

Integration of a reconstituted de novo synthesized hemoprotein and native metallo-proteins with electrode supports for bioelectronic and bioelectrocatalytic applications

J Am Chem Soc

Heme redox potential control in de novo designed four-alpha-helix bundle proteins

Biochemistry

Review
Engineering and design in the bioelectrochemistry of metalloproteins