De Novo Protein Design of Photochemical Reaction Centers

Abstract Natural photosynthetic protein complexes capture sunlight to power the energetic catalysis that supports life on Earth. Yet these natural protein structures carry an evolutionary legacy of complexity and fragility that encumbers protein reengineering efforts and obfuscates the underlying design rules for light-driven charge separation. De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build new enzymes for efficient solar-to-fuel energy conversion. Here we report the rational design, X-ray crystal structure, and electron transfer activity of a multi-cofactor protein that incorporates essential elements of photosynthetic reaction centers. This highly stable, modular artificial protein framework can be reconstituted in vitro with interchangeable redox centers for nanometer-scale photochemical charge separation. Transient absorption spectroscopy demonstrates Photosystem II-like tyrosine and metal cluster oxidation, and we measure charge separation lifetimes exceeding 100 ms, ideal for light-activated catalysis. This de novo-designed reaction center builds upon engineering guidelines established for charge separation in earlier synthetic photochemical triads and modified natural proteins, and it shows how synthetic biology may lead to a new generation of genetically encoded, light-powered catalysts for solar fuel production.

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Excitonic structure and charge separation in the heliobacterial reaction center probed by multispectral multidimensional spectroscopy

Nature Communications ◽

10.1038/s41467-021-23060-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yin Song ◽

Riley Sechrist ◽

Hoang H. Nguyen ◽

William Johnson ◽

Darius Abramavicius ◽

...

Keyword(s):

Reaction Center ◽

Charge Separation ◽

Electronic Spectroscopy ◽

Spectroscopic Studies ◽

Primary Electron ◽

Reaction Centers ◽

Separation Mechanism ◽

Photosynthetic Reaction Centers ◽

Density Analysis ◽

Function Relationship

AbstractPhotochemical reaction centers are the engines that drive photosynthesis. The reaction center from heliobacteria (HbRC) has been proposed to most closely resemble the common ancestor of photosynthetic reaction centers, motivating a detailed understanding of its structure-function relationship. The recent elucidation of the HbRC crystal structure motivates advanced spectroscopic studies of its excitonic structure and charge separation mechanism. We perform multispectral two-dimensional electronic spectroscopy of the HbRC and corresponding numerical simulations, resolving the electronic structure and testing and refining recent excitonic models. Through extensive examination of the kinetic data by lifetime density analysis and global target analysis, we reveal that charge separation proceeds via a single pathway in which the distinct A0 chlorophyll a pigment is the primary electron acceptor. In addition, we find strong delocalization of the charge separation intermediate. Our findings have general implications for the understanding of photosynthetic charge separation mechanisms, and how they might be tuned to achieve different functional goals.

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Excitonic Structure and Charge Separation in the Heliobacterial Reaction Center Probed by Multispectral Multidimensional Spectroscopy

10.26434/chemrxiv.13519169.v1 ◽

2021 ◽

Author(s):

Yin Song ◽

Riley Sechrist ◽

Hoang Huy Nguyen ◽

William Johnson ◽

Darius Abramavičius ◽

...

Keyword(s):

Reaction Center ◽

Charge Separation ◽

Electronic Spectroscopy ◽

Spectroscopic Studies ◽

Primary Electron ◽

Reaction Centers ◽

Separation Mechanism ◽

Photosynthetic Reaction Centers ◽

Density Analysis ◽

Function Relationship

Photochemical reaction centers are the engines that drive photosynthesis. The reaction center from heliobacteria (HbRC) has been proposed to most closely resemble the common ancestor of photosynthetic reaction centers, motivating a detailed understanding of its structure-function relationship. The recent elucidation of the HbRC crystal structure motivates advanced spectroscopic studies of its excitonic structure and charge separation mechanism. We perform multispectral two-dimensional electronic spectroscopy of the HbRC and corresponding numerical simulations, resolving the electronic structure and testing and refining recent excitonic models. Through extensive examination of the kinetic data by lifetime density analysis and global target analysis, we reveal that charge separation proceeds via a single pathway in which the distinct A0 chlorophyll a pigment is the primary electron acceptor. In addition, we find strong delocalization of the initial excited state and charge separation intermediate. Our findings have general implications for the understanding of photosynthetic charge separation mechanisms, and how they might be tuned to achieve different functional goals.

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The Methionine-aromatic Motif Plays a Unique Role in Stabilizing Protein Structure

Journal of Biological Chemistry ◽

10.1074/jbc.m112.374504 ◽

2012 ◽

Vol 287 (42) ◽

pp. 34979-34991 ◽

Cited By ~ 154

Author(s):

Christopher C. Valley ◽

Alessandro Cembran ◽

Jason D. Perlmutter ◽

Andrew K. Lewis ◽

Nicholas P. Labello ◽

...

Keyword(s):

Rational Design ◽

Protein Structures ◽

Data Bank ◽

Unique Role ◽

Hydrophobic Residues ◽

Receptor Complexes ◽

Affinity Ligand ◽

Additional Stabilization ◽

High Affinity Ligand

Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1–1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins.

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Constructing synthetic-protein assemblies from de novo designed 310 helices

10.1101/2021.12.11.471898 ◽

2021 ◽

Author(s):

Prasun Kumar ◽

Neil G. Paterson ◽

Jonathan Clayden ◽

Derek N. Woolfson

Keyword(s):

Rational Design ◽

De Novo ◽

Protein Structures ◽

Water Soluble ◽

Design Solution ◽

Protein Assemblies ◽

Synthetic Protein ◽

Hydrophobic Cores ◽

Α Helix ◽

Helical Folding

Compared with the iconic α helix, 310 helices occur much less frequently in protein structures. The different 310-helical parameters lead to energetically less favourable internal energies, and a reduced tendency to pack into defined higher-order structures. Consequently, in natural proteins, 310 helices rarely extend past 6 residues, and do not form regular supersecondary, tertiary, or quaternary interactions. Here, we show that despite their absence in nature, synthetic protein-like assemblies can be built from 310 helices. We report the rational design, solution-phase characterisation, and an X-ray crystal structure for water-soluble bundles of 310 helices with consolidated hydrophobic cores. The design uses 6-residue repeats informed by analysing natural 310 helices, and incorporates aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilising assemblies in this unexplored region of protein-structure space.

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Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms222413451 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13451

Author(s):

Anna M. Banaś ◽

Katarzyna M. Bocian-Ostrzycka ◽

Stanisław Dunin-Horkawicz ◽

Jan Ludwiczak ◽

Piotr Wilk ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Protein Structures ◽

Essential Elements ◽

Bacterial Physiology ◽

Bacterial Proteins ◽

In Vivo Experiments ◽

Redox Partner ◽

Periplasmic Transport

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

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Primary processes in the bacterial reaction center probed by two-dimensional electronic spectroscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721927115 ◽

2018 ◽

Vol 115 (14) ◽

pp. 3563-3568 ◽

Cited By ~ 20

Author(s):

Andrew Niedringhaus ◽

Veronica R. Policht ◽

Riley Sechrist ◽

Arkaprabha Konar ◽

Philip D. Laible ◽

...

Keyword(s):

Excited State ◽

Reaction Center ◽

Charge Separation ◽

Purple Bacteria ◽

Electronic Spectroscopy ◽

Reaction Centers ◽

Purple Bacterium ◽

Absorption Bands ◽

Sequential Reaction ◽

Special Pair

In the initial steps of photosynthesis, reaction centers convert solar energy to stable charge-separated states with near-unity quantum efficiency. The reaction center from purple bacteria remains an important model system for probing the structure–function relationship and understanding mechanisms of photosynthetic charge separation. Here we perform 2D electronic spectroscopy (2DES) on bacterial reaction centers (BRCs) from two mutants of the purple bacterium Rhodobacter capsulatus, spanning the Qy absorption bands of the BRC. We analyze the 2DES data using a multiexcitation global-fitting approach that employs a common set of basis spectra for all excitation frequencies, incorporating inputs from the linear absorption spectrum and the BRC structure. We extract the exciton energies, resolving the previously hidden upper exciton state of the special pair. We show that the time-dependent 2DES data are well-represented by a two-step sequential reaction scheme in which charge separation proceeds from the excited state of the special pair (P*) to P+HA− via the intermediate P+BA−. When inhomogeneous broadening and Stark shifts of the B* band are taken into account we can adequately describe the 2DES data without the need to introduce a second charge-separation pathway originating from the excited state of the monomeric bacteriochlorophyll BA*.

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Excitonic Structure and Charge Separation in the Heliobacterial Reaction Center Probed by Multispectral Multidimensional Spectroscopy

10.26434/chemrxiv.13519169 ◽

2021 ◽

Author(s):

Yin Song ◽

Riley Sechrist ◽

Hoang Huy Nguyen ◽

William Johnson ◽

Darius Abramavičius ◽

...

Keyword(s):

Reaction Center ◽

Charge Separation ◽

Electronic Spectroscopy ◽

Spectroscopic Studies ◽

Primary Electron ◽

Reaction Centers ◽

Separation Mechanism ◽

Photosynthetic Reaction Centers ◽

Density Analysis ◽

Function Relationship

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De novo protein design, a retrospective

Quarterly Reviews of Biophysics ◽

10.1017/s0033583519000131 ◽

2020 ◽

Vol 53 ◽

Cited By ~ 20

Author(s):

Ivan V. Korendovych ◽

William F. DeGrado

Keyword(s):

Membrane Protein ◽

Protein Design ◽

First Principles ◽

Rational Design ◽

De Novo ◽

Dynamic Properties ◽

Protein Structures ◽

Amino Acid Sequences ◽

Physical Models ◽

And Function

Abstract Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic polymers that function in mixed or non-aqueous environments, is now becoming increasingly possible.

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Computational design of a modular protein sense-response system

Science ◽

10.1126/science.aax8780 ◽

2019 ◽

Vol 366 (6468) ◽

pp. 1024-1028 ◽

Cited By ~ 12

Author(s):

Anum A. Glasgow ◽

Yao-Ming Huang ◽

Daniel J. Mandell ◽

Michael Thompson ◽

Ryan Ritterson ◽

...

Keyword(s):

De Novo ◽

Protein Structures ◽

Computational Design ◽

Metabolic Intermediate ◽

Protein Interfaces ◽

Substantial Progress ◽

Valuable Compounds ◽

Protein Sensors ◽

New Protein

Sensing and responding to signals is a fundamental ability of living systems, but despite substantial progress in the computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here, we describe a generalizable computational strategy for designing sensor-actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation through split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site closely matches the design model. Our computational design strategy opens broad avenues to link biological outputs to new signals.

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Computational design of a modular protein sense/response system

10.1101/648485 ◽

2019 ◽

Author(s):

Anum A. Glasgow ◽

Yao-Ming Huang ◽

Daniel J. Mandell ◽

Michael Thompson ◽

Ryan Ritterson ◽

...

Keyword(s):

De Novo ◽

Protein Structures ◽

Computational Design ◽

Metabolic Intermediate ◽

Signaling Systems ◽

Engineering Strategy ◽

Valuable Compounds ◽

Protein Sensors ◽

New Protein

ABSTRACTSensing and responding to signals is a fundamental ability of living systems, but despite remarkable progress in computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here we describe a generalizable computational strategy for designing sensor/actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation via split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site matches the design model with atomic accuracy. Our computational design strategy opens broad avenues to link biological outputs to new signals.One Sentence SummaryAn engineering strategy to design modular synthetic signaling systems that respond to new small molecule inputs.

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