Metalloprotein Design and Engineering

Chakraborty, Saumen; Hosseinzadeh, Parisa; Lu, Yi

doi:10.1002/9781119951438.eibc0259.pub2

Cited by 7 publications

(10 citation statements)

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“…In this section, we provide an overview of the efforts on designing metalloproteins as ET centers, as well as designing artificial networks and pathways involving metalloproteins. More in-depth discussions are summarized in other reviews in this field [51–55]. …”

Section: Design Of Et Centersmentioning

confidence: 99%

Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics

Hosseinzadeh¹,

Lu²

2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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121

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Redox potentials are the major contributors to controlling the ET rates and thus regulating ET processes in the bioenergetics. To maximize the efficiency of the ET process, one needs to master the art of tuning of the E°, especially metalloproteins, as they represent major classes of ET proteins. In this review, we first describe the importance of tuning E° of ET centers, including the metalloproteins described above, and its role in regulating the ET in bioenergetic processes including photosynthesis and respiration. The major focus of this review is to summarize recent work in designing the ET centers, namely cupredoxins, cytochromes, and iron-sulfur proteins, and examples in design of protein networks involved these ET centers. We then discuss the factors that affect redox potentials of these ET centers including metal ion, the ligands to metal center and interactions beyond the primary ligand, especially non-covalent secondary coordination sphere interactions. We gave examples of strategies to fine-tune the redox potential using both natural and unnatural amino acids and native and nonnative cofactors. Several case studies are used to illustrate recent successes in this area. Outlooks for future endeavors are also provided.

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Section: Design Of Et Centersmentioning

confidence: 99%

Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics

Hosseinzadeh¹,

Lu²

2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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162

121

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“…General purification of our designed heteronuclear metalloproteins will not be discussed in great detail here, but generally falls into two categories: inclusion body and cytosolic purification. The inclusion body protocol is used extensively in models such as Cu B Mb and Fe B Mb and has been covered previously in those works (Springer & Sligar, 1987, Sigman et al, 2000, Sigman et al, 2000, Sigman et al, 2003, Pfister et al, 2005, Zhao et al, 2005, Zhao et al, 2006, Yeung et al, 2009, Lin et al, 2010, Lin et al, 2010, Miner et al, 2012, Chakraborty et al, 2014, Bhagi-Damodaran et al, 2014, Petrik et al, 2016). Additionally, cytosolic purification of C c P and its rationally designed mutants has also been described in great detail by our group (Yeung et al, 1997, Sigman et al, 1999, Feng et al, 2003, Pfister et al, 2007, Miner et al, 2014, Hosseinzadeh et al, 2016).…”

Section: Step 2: Purification and Structural Characterization Of Ratimentioning

confidence: 99%

“…To overcome this challenge, the heme can be replaced with Zn protoporphyrin IX. This replacement has been successfully done in Fe B -Mb protein for the complete characterization of nonhemeFe(II)-NO species via UV-Vis, EPR, and Mössbauer spectroscopy (Chakraborty et al, 2014). …”

Section: Step 3: Functional Characterization Of Designed Heterobinuclmentioning

confidence: 99%

“…Figure 4a and 4b is adapted with permission from ref. (Chakraborty et al, 2014) and (Lin et al, 2010) respectively.…”

Section: Figurementioning

confidence: 99%

“…To complement the study of native enzymes and synthetic models, we and others have employed a biosynthetic approach of designing heteronuclear metalloenzymes using small, stable scaffold proteins that can already be expressed in high yields with exceptional purity through well-established purification schemes (DeGrado et al, 1989, DeGrado et al, 1999, Gibney et al, 1999, Lu et al, 1999, Reedy & Gibney, 2004, Thomas & Ward, 2005, Zanghellini et al, 2006, Lu et al, 2009, Lu et al, 2009, Richter et al, 2011, Zastrow & Pecoraro, 2013, Lu et al, 2013, Marshall et al, 2013, Chakraborty et al, 2014). Protein scaffolds are innately optimized to function under physiological conditions, offer a broad ligand set that can be expanded through the use of non-canonical amino acids, and comprise a genetically-programmable platform for bottom-up engineering of metal binding sites.…”

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Design of Heteronuclear Metalloenzymes

Bhagi‐Damodaran

Hosseinzadeh

Mirts

et al. 2016

Methods in Enzymology

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Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme-nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedure on how to choose the protein scaffold, design a heterobinuclear metal center in the protein computationally, incorporate metal centers in the protein and characterize the resulting metalloprotein, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity.

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Nanosecond Dynamics at Protein Metal Sites: An Application of Perturbed Angular Correlation (PAC) of γ-Rays Spectroscopy

et al. 2017

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Metalloproteins are essential to numerous reactions in nature, and constitute approximately one-third of all known proteins. Molecular dynamics of proteins has been elucidated with great success both by experimental and theoretical methods, revealing atomic level details of function involving the organic constituents on a broad spectrum of time scales. However, the characterization of dynamics at biomolecular metal sites on nanosecond time scales is scarce in the literature. The aqua ions of many biologically relevant metal ions exhibit exchange of water molecules on the nanosecond time scale or faster, often defining their reactivity in aqueous solution, and this is presumably also a relevant time scale for the making and breaking of coordination bonds between metal ions and ligands at protein metal sites. Ligand exchange dynamics is critical for a variety of elementary steps of reactions in metallobiochemistry, for example, association and dissociation of metal bound water, association of substrate and dissociation of product in the catalytic cycle of metalloenzymes, at regulatory metal sites which require binding and dissociation of metal ions, as well as in the transport of metal ions across cell membranes or between proteins involved in metal ion homeostasis. In Perturbed Angular Correlation of γ-rays (PAC) spectroscopy, the correlation in time and space of two γ-rays emitted successively in a nuclear decay is recorded, reflecting the hyperfine interactions of the PAC probe nucleus with the surroundings. This allows for characterization of molecular and electronic structure as well as nanosecond dynamics at the PAC probe binding site. Herein, selected examples describing the application of PAC spectroscopy in probing the dynamics at protein metal sites are presented, including (1) exchange of Cd bound water in de novo designed synthetic proteins, and the effect of remote mutations on metal site dynamics; (2) dynamics at the β-lactamase active site, where the metal ion appears to jump between the two adjacent sites; (3) structural relaxation in small blue copper proteins upon Ag to Cd transformation in radioactive nuclear decay; (4) metal ion transfer between two HAH1 proteins with change in coordination number; and (5) metal ion sensor proteins with two coexisting metal site structures. With this Account, we hope to make our modest contribution to the field and perhaps spur additional interest in dynamics at protein metal sites, which we consider to be severely underexplored. Relatively little is known about detailed atomic motions at metal sites, for example, how ligand exchange processes affect protein function, and how the amino acid composition of the protein may control this facet of metal site characteristics. We also aim to provide the reader with a qualitative impression of the possibilities offered by PAC spectroscopy in bioinorganic chemistry, especially when elucidating dynamics at protein metal sites, and finally present data that may serve as benchmarks on a relevant time scale for development a...

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Metalloprotein Design and Engineering

Cited by 7 publications

References 478 publications

Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics

Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics

Design of Heteronuclear Metalloenzymes

Nanosecond Dynamics at Protein Metal Sites: An Application of Perturbed Angular Correlation (PAC) of γ-Rays Spectroscopy

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