Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane α-helical barrels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) a low-conductance states that correlate with variations in the modeled coiled-coil barrel geometries, indicating stable transmembrane α-helical barrels; and (ii) high-conductance states in which single pores change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic pores through the recruitment and expulsion of peptides, as observed in natural barrel-stave peptide pores. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry. ### Competing Interest Statement The authors have declared no competing interest.

Biotechnology and BioengineeringVolume 114, Issue 6 p. 1123-1127 Issue Information - TOCFree Access Biotechnology and Bioengineering: Volume 114, Number 6, June 2017 First published: 20 April 2017 https://doi.org/10.1002/bit.26136AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume114, Issue6June 2017Pages 1123-1127 RelatedInformation

The iron-sulfur cluster containing protein Fumarate and Nitrate Reduction (FNR) is the master regulator for the switch between anaerobic and aerobic respiration in Escherichia coli and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O-2 that leads to conversion to a [2Fe-2S] form with loss of high-affinity DNA binding. Here, we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a [3Fe-3S] cluster and persulfide-coordinated [2Fe-2S] clusters [[2Fe-2S](S)(n), where n = 1 or 2]. Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion and not as a subsequent, secondary reaction to generate [2Fe-2S](S)(n) species. This methodology shows great potential for broad application to studies of protein cofactor-small molecule interactions.

AbstractThe reaction of protein‐bound iron–sulfur (Fe‐S) clusters with nitric oxide (NO) plays key roles in NO‐mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe‐S clusters has been hampered by a lack of information about the nature of the iron‐nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe‐4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2(NO)4(Cys)2]) and Roussin's Black Salt (RBS, [Fe4(NO)7S3]. In the latter case, the absence of 32S/34S shifts in the Fe−S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions.

Robert J. P. Williams was a pioneer in advancing our understanding of the roles of chemical elements, especially the metals, in biology and in biological evolution. During the first half of his career of more than 60 years at Oxford University he studied the thermodynamic stabilities of transition-metal complexes with organic ligands, their redox properties, magnetism and colour, to understand their biological function. In parallel he collaborated with biologists and biophysicists, for example with Bert Vallee, studying zinc in proteins. Williams was the first to describe how proton gradients could be used to drive the formation of the universal biological fuel, ATP (adenosine triphosphate), a fundamental step in biological energetics. From the late 1960s he studied many proteins that use metal ions for catalysis, for electron transfer and cellular regulation. A leading figure in the establishment of the Oxford Enzyme Group, Williams developed high-field nuclear magnetic resonance (NMR) to study the mobility and dynamics of many protein structures, leading to a deeper understanding of protein function. He held the Royal Society Napier Research Professorship from 1974 until his retirement in 1991. Subsequently he published several books setting out his understanding of the roles of metal ions in biology, and their wider significance in evolution. Bob Williams's deep insights across many disciplines made him a charismatic teacher. His lateral style of thinking never failed to inspire. His legacy lies in the successful careers of his many students and collaborators worldwide and the vigour of the new discipline of bioinorganic chemistry that he helped to establish.

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe–4S] cluster-containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe–4S] to [2Fe–2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~sixfold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer–dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe–4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers.

NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrR promoters, ∼4 and ∼8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8-10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ∼2 NO per cluster, along with two further intermediates at ∼4 and ∼6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes.

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O-2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O-2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O-2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O-2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O-2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O-2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O-2.

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR.

Robert (Bob) J. P. Williams who, after a short illness, died on the 21st March 2015 at the age of 89, was professor emeritus at the University of Oxford and was internationally recognized as a founding father of bioinorganic chemistry. His pioneering studies on the roles of metal ions in biological systems brought unprecedented insights into the structure, function, and dynamics of metalloproteins, and established a better understanding of biological signaling, electron-transfer processes, and enzyme catalysis. As one of the inaugural members of the Oxford Enzyme Group in 1972, he employed new developments in high-field NMR spectroscopy to reveal the connection between conformational change and biological function in iron- and calcium-binding proteins, cytochromes, and kinases. In the latter part of his career, Williams initiated a new area of study elucidating the chemical principles responsible for the structure, morphology, and function of calcium carbonate, silica, and iron oxide biominerals. Williams studied chemistry at Merton College, Oxford for both his BA and DPhil (1944–1950). During his undergraduate research project, he published, with Harry M. N. H. Irving, a seminal paper in Nature (1948) on the relative stabilities of metal-ion transition-metal complexes, that became widely known as the Irving–Williams series. After Oxford, Williams took up a one-year postdoctoral position with Arne Tiselius in Uppsala, Sweden, where he developed chromatographic techniques for protein isolation, and published, as sole author, a ground-breaking paper in Biological Reviews (1953) entitled “Metal ions in biological systems”. There he also met Jelly Büchli, whom he married in 1952. He returned to Merton College as a Junior Research Fellow (1951–1955), and was appointed in 1955 as Tutor in Chemistry at Wadham College and University Lecturer in the Inorganic Chemistry Laboratory. He subsequently switched to teaching biochemistry to undergraduates to widen his knowledge. Williams took sabbatical leave at Harvard Medical School in 1966 to work with Bert Vallee, which led to a long collaboration on the isolation of the Zn carboxypeptidase and the replacement of the ZnII ion with the spectroscopically active CoII ion. After his retirement in 1995 he held emeritus positions at Oxford. Bob’s long, outstanding career has been recognized by numerous international honors. He was elected as a Fellow of the Royal Society in 1972, and was a member of four further national academies, as well as the recipient of several honorary degrees. He was also recognized with many distinguished awards and titled lectures. He published over 700 articles, including several highly original books offering novel insights on general inorganic chemistry, the inorganic chemistry of life, the natural selection of the elements, and most recently, the co-evolution of the chemistry of the environment and life (such as The Biological Chemistry of the Elements—The Inorganic Chemistry of Life (with J. J. R. Fausto); and Evolution’s Destiny: Co-evolving Chemistry of the Environment and Life (with R. Rickaby)). In recognition of his distinguished career, Wadham College established no less than three academic positions that bear his name. Bob was awarded an MBE (Member of the Most Excellent Order of the British Empire) in 2010 in recognition of his contributions to his local community in Oxford. Bob Williams was a gifted man, wonderfully creative, highly inspirational, a brilliant teacher and supervisor, and passionate about many areas of science. He was a true polymath, with an encyclopedic knowledge of chemistry, biochemistry and geochemistry. With a sharp mind and fertile imagination he always sought to discover the larger picture and underlying principles hidden behind the facts. Bob was generous in sharing his insights sometimes before there was strong experimental evidence. Others were not slow to seize on his ideas. In 1960, he wrote papers describing a mechanism for the long-range conversion of the energy of reaction of oxygen and hydrogen into localized proton gradients and connecting this coupling to ATP production. Thus began an interest in the chemical processes of respiration and an examination of the possible roles of chains of catalysts within mitochondrial membranes. These ideas contrasted sharply with those of biochemists who were hunting for high-energy phosphorylated intermediates. After a lengthy correspondence with Williams, P. D. Mitchell took up the essence of these ideas to formulate his chemiosmotic hypothesis. Later, Williams developed a deep interest in how chemical constraints were fundamental to the evolution of life. Williams’ legacy to chemistry and biochemistry is enormous. His influence lives on amongst a widespread cadre of scientists beyond those who worked directly with him. No fewer than six of his former students have been elected to the Royal Society. Bob had a lively and engaging personality, and was generous and considerate to his students. The affection and loyalty felt by many extends across the world. Bob Williams will be sadly missed.

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.