Ville R. I. Kaila

Complex I is a large membrane protein that transduces redox energy into a proton motive force across a biological membrane, powering cellular respiration. Yet, despite significant experimental and computational efforts, its long-range proton translocation mechanism remains poorly understood. Here, we combine nuclear magnetic resonance spectroscopy and site-directed mutagenesis experiments with multiscale classical and hybrid quantum/classical (QM/MM) simulations to probe how conformational dynamics is linked to proton transfer reactions in the terminal antiporter module (Nqo12/NuoL/ND5) of Complex I. We show that protonation of a conserved proton-loading site (Lys329/His241 ^Nqo12 ) in the terminal antiporter-like module gives rise to conformational changes that result in opening of the proton pathway. We further show that this protonation reaction is controlled by buried ion-pairs that modulate the barrier for proton translocation by intrinsic electric field effects. On a general level, our combined findings reveal how bioenergetic machines combine conformational dynamics with hydration transitions to establish efficient redox-driven proton pumps.

Ion-pairs are central charged motifs that control the biological function of several proteins. Yet, the physical principles linked to the formation of ion-pairs during protein folding remain poorly understood. Here, we show that buried ion-pairs of de novo -designed helical bundle proteins reshape the folding pathways by introducing kinetic barriers that are overcome by a hydrophobic collapse linked to packing of the protein core. By combining protein design with co-translational folding studies, nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics simulations, we show that introduction of a buried ion-pair leads to a shifted folding transition that balances desolvation and electrostatic interactions within the protein and the bulk solvent. Moreover, we find that the folding pathway is sensitive to modifications in non-polar interactions and motifs that electrostatically shield the ion-pair from its surroundings. Our combined findings provide a blueprint for understanding how functional motifs affect protein folding and how mutation of charged networks alter the stability of proteins. Significance Statement: Despite significant progress in machine learning-based structure prediction, we still lack a clear understanding of the folding pathways that determine how proteins fold into their exact three-dimensional structure. Here, we show that introduction of a buried ion-pair reshapes the folding pathway of de novo -designed protein Maquettes . By combining cotranslocation folding experiments of these proteins trapped within the ribosome exit tunnel with chemical unfolding experiments using nuclear magnetic resonance spectroscopy and atomistic simulations, we show that the ion-pair introduces a folding barrier linked to formation of the protein core. On a general level, our findings highlight how charged networks balance electrostatic and solvation forces in proteins, and how mutations can alter their folding landscape.

Complex I is a highly intricate membrane-bound protein complex that powers the cellular energy metabolism by a long-range ( > 300 Å) proton-coupled electron transfer (PCET) reaction. Here, we investigate the highly debated coupling mechanism of Complex I by probing the charge transfer reaction along its functionally central carboxylate pathway (E-channel). By combining biophysical and site-directed mutagenesis experiments with high-resolution (2.6-2.8 Å) cryo-electron microscopy (cryo-EM) and multiscale simulations, we identify a conserved carboxylate switch point (D79 ^NuoA ) that mediates proton transfer by establishing a kinetic gate and couples the redox chemistry to proton pumping. We find that mutation of the identified site, as found in patients suffering from severe neurodegenerative disorders, drastically perturbs the charge transfer mechanism, and results in a 20% PCET activity. Our combined findings illustrate mechanistic principles of molecular gates underlying long-range charge transfer reactions, and show how disease mutations perturb the function of conserved switch points in energy transduction.

Site-direct mutagenesis provides a basis for functional studies of proteins. Yet, despite many available techniques, mutagenesis of large protein complexes can be tedious and highly challenging, with the replication fidelity and long-range amplification limiting the process. Here, we develop a method for site-directed mutagenesis of large protein complexes based on sub-fragmentation of its coding sequence that generates libraries of shorter sequences, where the DNA polymerase can accurately catalyse long-range elongation. Using the nuo genes encoding for the 0.5 MDa E. coli Complex I, we successfully dissect the 15.1 kb long DNA sequence into fragments of 900 bp with partially overlapping regions and unique primer sequences, creating a plasmid library that can be amplified by regular DNA polymerases. We showcase the method by sub-cloning the E. coli Complex I sequence into 20 fragments from a large pBAD expression vector (21.3 kb). The method shows a high success rate of introduced mutations, and an efficient Gibson assembly of the mutated fragments into the expression vector, enabling the accurate introduction of point-mutations into the large pBAD vector. We suggest that the plasmid sub-fragmentation provides an efficient method for mutagenesis of multi-subunit protein complexes with several homologous subunits, such as the respiratory Complex I, advancing mechanistic studies of large bioenergetics protein complexes.

Phosphorylation is among the most ubiquitous and essential posttranslational modifications in biological systems. It is regulated by highly complex enzymatic networks. Here, we explore enzyme-free phosphorylation in a designed peptide system. Specifically, we use phosphorylation to modulate coiled-coil (CC) assembly and dynamics. This exploits a nonbiological reaction cycle to phosphorylate and dephosphorylate a histidine residue in an α-helix, enabling or disabling CC formation with a second helix. Dephosphorylation is kinetically gated─it is 25× faster in the CC compared to the nonassembled state. As a result, the cycle of phosphorylation, CC formation, dephosphorylation, and CC disassembly is ratcheted. The minimal synthetic phosphorylation cycle provides design principles and fulfills requirements to perform work mimicking the function of molecular motors, walkers, and pumps.

The respiratory Complex I is a highly intricate redox-driven proton pump that powers oxidative phosphorylation across all domains of life. Yet, despite major efforts, its long-range energy transduction principles remain much debated. Here, we study the molecular principles of proton transport by engineering the antiporter modules of Complex I. By combining directed mutagenesis with time-resolved spectroscopy and molecular dynamics (MD) simulations, we identify conserved residues along the proton channels that control the rate of proton transfer across proteoliposome membranes. The antiporter modules catalyze this tightly regulated proton transport by transient water wires that follow intrinsic electric fields along the proton channels. Based on MD simulations, we identify conserved gating sites, established by nonpolar residues, which modulate the hydration and electric field effects underlying the proton transport upon mutation. On a general level, our findings highlight how the modular energy-transduction machinery of Complex I employs a combination of electrostatic and conformational coupling principles to catalyze long-range proton transport, with distinct similarities to other enzymes.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 Å) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combining multiscale quantum and classical simulations with directed mutagenesis, X-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable 3,4-dihydroxyphenylalanine (DOPA) initiator radical. We show that DOPA• is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis.

Complex I is a gigantic redox-driven proton pump that powers oxidative phosphorylation by a unique long-range (>200 Å) proton-coupled electron transfer process. To elucidate the molecular principles underlying this intricate action-at-a-distance effect, we combine here multiscale quantum/classical (QM/MM) simulations with site-directed mutagenesis, proteoliposome experiments, and cryo-electron microscopy (cryo-EM). We find that quinol binding at a distinct site within a local membrane cavity, triggers a long-range protonation cascade over water-mediated proton wires along a conserved carboxylate pathway (E-channel). We identify a central mechanical switch point, comprising the conserved Tyr156 ^H , the mutation of which impedes conformational changes along conserved loops, but not the proton transfer reaction itself. Using our integrative multi-disciplinary approach, we reveal central coupling sites along the redox-driven proton transport process mediating energy conversion in Complex I, and illustrate the power of theory-guided experiments.

The bioenergetic complexes of energy-transducing membranes generate a proton current that powers the synthesis of adenosine triphosphate. Yet, since the early days of the chemiosmotic theory, it has remained elusive and much debated whether the proton motive force (PMF) delocalizes into the bulk solvent surrounding the energy-transducing membrane or if the thermodynamic force is exerted as a localized proton current along the membrane surface. To elucidate the molecular principles underlying protonation dynamics at biological membranes, we combine here proteoliposome experiments with fluorescence correlation spectroscopy and multiscale molecular simulations. We show that ubiquinone (Q ₁₀ ), which is an essential electron carrier of inner mitochondrial membranes, interacts with protons at the membrane, and alters the rate of the protonation reactions along the surface. We find that physiological Q ₁₀ concentrations increase the integrity of the liposome membranes to sustain a PMF and enhance the rate of surface protonation reactions of lipid-conjugated pH-sensitive fluorophores, occurring on a microsecond timescale. Our multiscale simulations reveal that the quinone headgroup localizes at the membrane surface and stabilizes protonated water species by cation–π and hydrogen-bonded interactions amplifying the proton exchange on the surface relative to the bulk solvent. We suggest that in addition to the well-established role of quinones as redox mediators in energy-transducing membranes, Q ₁₀ also promotes the proton-collecting antenna effect, mediating proton exchange along the membrane and supporting a local proton circuit model. Our combined findings provide molecular insight into propagation of proton currents along biological membranes and reveal key principles underlying the energy conversion mechanisms in biology.

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here, we characterize the cyt bd -II from Mycobacterium smegmatis , a member of a hitherto uncharacterized evolutionary group (qOR-2) of bd oxidases, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. Overexpressing the appCB operon in its native host led to production of a highly active bd -II ( k _obs = 30 e ⁻ s ⁻¹ ) that together with a high-resolution (2.8 Å) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.