Search

Experimental NMR spectroscopy and theoretical molecular dynamics (MD) simulations provide complementary insights into protein conformational dynamics and hence into biological function. The present work describes an extensive set of backbone NH and side-chain methyl group generalized order parameters for the Escherichia coli ribonuclease HI (RNH) enzyme derived from 2-μs microsecond MD simulations using the OPLS4 and AMBER-FF19SB force fields. The simulated generalized order parameters are compared with values derived from NMR 15 N and 13 CH 2 D spin relaxation measurements. The squares of the generalized order parameters, S 2 for the N-H bond vector and S axis 2 for the methyl group symmetry axis, characterize the equilibrium distribution of vector orientations in a molecular frame of reference. Optimal agreement between simulated and experimental results was obtained by averaging S 2 or S axis 2 calculated by dividing the simulated trajectories into 50 ns blocks (∼five times the rotational diffusion correlation time for RNH). With this procedure, the median absolute deviations (MAD) between experimental and simulated values of S 2 and S axis 2 are 0.030 (NH) and 0.061 (CH 3 ) for OPLS4 and 0.041 (NH) and 0.078 (CH 3 ) for AMBER-FF19SB. The MAD between OPLS4 and AMBER-FF19SB are 0.021 (NH) and 0.072 (CH 3 ). The generalized order parameters for the methyl group symmetry axis can be decomposed into contributions from backbone fluctuations, between-rotamer dihedral angle transitions, and within-rotamer dihedral angle fluctuations. Analysis of the simulation trajectories shows that ( i ) backbone and side chain conformational fluctuations exhibit little correlation and that ( ii ) fluctuations within rotamers are limited and highly uniform with values that depend on the number of dihedral angles considered. Low values of S axis 2 , indicative of enhanced side-chain flexibility, result from between-rotamer transitions that can be enhanced by increased local backbone flexibility.

Background: The study explores whether frailty at midlife predicts mortality and levels of biomarkers associated with Alzheimer's disease and related dementias (ADRD) and neurodegeneration by early old age. We also examine the heritability of frailty across this age period., Methods: Participants were 1,286 community-dwelling men from the Vietnam Era Twin Study of Aging at average ages 56, 62 and 68, all without ADRD at baseline. The cumulative deficit frailty index (FI) comprised 37 items assessing multiple physiological systems. Plasma biomarkers at age 68 included beta-amyloid (Aβ40, Aβ42), total tau (t-tau) and neurofilament light chain (NfL)., Results: Being frail doubled the risk of all-cause mortality by age 68 (OR = 2.44). Age 56 FI significantly predicted age 68 NfL (P = 0.014), Aβ40 (P = 0.001) and Aβ42 (P = 0.023), but not t-tau. Age 62 FI predicted all biomarkers at age 68: NfL (P = 0.023), Aβ40 (P = 0.002), Aβ42 (P = 0.001) and t-tau (P = 0.001). Age 68 FI scores were associated with age 68 levels of NfL (P = 0.027), Aβ40 (P < 0.001), Aβ42 (P = 0.001) and t-tau (P = 0.003). Genetic influences accounted for 45-48% of the variance in frailty and significantly contributed to its stability across 11 years., Conclusions: Frailty during one's 50s doubled the risk of mortality by age 68. A mechanism linking frailty and ADRD may be through its associations with biomarkers related to neurodegeneration. Cumulative deficit frailty increases with age but remains moderately heritable across the age range studied. With environmental factors accounting for about half of its variance, early interventions aimed at reducing frailty may help to reduce risk for ADRD., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Structural genomics consortia established that protein crystallization is the primary obstacle to structure determination using x-ray crystallography. We previously demonstrated that crystallization propensity is systematically related to primary sequence, and we subsequently performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. This software can be run interactively on the worldwide web at https://www.pxengineering.org/. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization, and, for two of these three proteins, the construct with the largest number of KR substitutions exhibits significantly enhanced crystallization propensity. This approach rapidly produced a 1.9 Å crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from Bulk KR-substituted domains show the engineered arginine residues frequently make hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that Bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve crystallization., (© 2024 The Protein Society.)

The evolutionary benefit accounting for widespread conservation of oligomeric structures in proteins lacking evidence of intersubunit cooperativity remains unclear. Here, crystal and cryo-EM structures, and enzymological data, demonstrate that a conserved tetramer interface maintains the active-site structure in one such class of proteins, the short-chain dehydrogenase/reductase (SDR) superfamily. Phylogenetic comparisons support a significantly longer polypeptide being required to maintain an equivalent active-site structure in the context of a single subunit. Oligomerization therefore enhances evolutionary fitness by reducing the metabolic cost of enzyme biosynthesis. The large surface area of the structure-stabilizing oligomeric interface yields a synergistic gain in fitness by increasing tolerance to activity-enhancing yet destabilizing mutations. We demonstrate that two paralogous SDR superfamily enzymes with different specificities can form mixed heterotetramers that combine their individual enzymological properties. This suggests that oligomerization can also diversify the functions generated by a given metabolic investment, enhancing the fitness advantage provided by this architectural strategy., (© 2022 The Authors.)

GPR158 is an orphan G protein–coupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryo–electron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gβ5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.

Cystic Fibrosis (CF) is caused by mutations to the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. CFTR is composed of two membrane spanning domains, two cytosolic nucleotide-binding domains (NBD1 and NBD2) and a largely unstructured R-domain. Multiple CF-causing mutations reside in the NBDs and some are known to compromise the stability of these domains. The ability to predict the effect of mutations on the stability of the cytosolic domains of CFTR and to shed light on the mechanisms by which they exert their effect is therefore important in CF research. With this in mind, we have predicted the effect on domain stability of 59 mutations in NBD1 and NBD2 using 15 different algorithms and evaluated their performances via comparison to experimental data using several metrics including the correct classification rate (CCR), and the squared Pearson correlation ( R 2 ) and Spearman's correlation (ρ) calculated between the experimental Δ T m values and the computationally predicted ΔΔ G values. Overall, the best results were obtained with FoldX and Rosetta. For NBD1 (35 mutations), FoldX provided R 2 and ρ values of 0.64 and -0.71, respectively, with an 86% correct classification rate (CCR). For NBD2 (24 mutations), FoldX R 2 , ρ, and CCR were 0.51, -0.73, and 75%, respectively. Application of the Rosetta high-resolution protocol (Rosetta&#95;hrp) to NBD1 yielded R 2 , ρ, and CCR of 0.64, -0.75, and 69%, respectively, and for NBD2 yielded R 2 , ρ, and CCR of 0.29, -0.27, and 50%, respectively. The corresponding numbers for the Rosetta's low-resolution protocol (Rosetta&#95;lrp) were R 2 = 0.47, ρ = -0.69, and CCR = 69% for NBD1 and R 2 = 0.27, ρ = -0.24, and CCR = 63% for NBD2. For NBD1, both algorithms suggest that destabilizing mutations suffer from destabilizing vdW clashes, whereas stabilizing mutations benefit from favorable H-bond interactions. Two triple consensus approaches based on FoldX, Rosetta&#95;lpr, and Rosetta&#95;hpr were attempted using either "majority-voting" or "all-voting". The all-voting consensus outperformed the individual predictors, albeit on a smaller data set. In summary, our results suggest that the effect of mutations on the stability of CFTR's NBDs could be largely predicted. Since NBDs are common to all ABC transporters, these results may find use in predicting the effect and mechanism of the action of multiple disease-causing mutations in other proteins.

Energy-dependent translational throttle A (EttA) from Escherichia coli is a paradigmatic ABC-F protein that controls the first step in polypeptide elongation on the ribosome according to the cellular energy status. Biochemical and structural studies have established that ABC-F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but not in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes, ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. Here, we review the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC-F proteins, and discuss the implications of these studies for the molecular function of eukaryotic ABC-F proteins, including the three human family members., (© 2020 Federation of European Biochemical Societies.)

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival., Competing Interests: Conflict of interest G. T. M. is the founder of Nexomics Biosciences Inc. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In Alzheimer's disease (AD), neurodegenerative processes are ongoing for years prior to the time that cortical atrophy can be reliably detected using conventional neuroimaging techniques. Recent advances in diffusion-weighted imaging have provided new techniques to study neural microstructure, which may provide additional information regarding neurodegeneration. In this study, we used neurite orientation dispersion and density imaging (NODDI), a multi-compartment diffusion model, in order to investigate cortical microstructure along the clinical continuum of mild cognitive impairment (MCI) and AD dementia. Using gray matter-based spatial statistics (GBSS), we demonstrated that neurite density index (NDI) was significantly lower throughout temporal and parietal cortical regions in MCI, while both NDI and orientation dispersion index (ODI) were lower throughout parietal, temporal, and frontal regions in AD dementia. In follow-up ROI analyses comparing microstructure and cortical thickness (derived from T1-weighted MRI) within the same brain regions, differences in NODDI metrics remained, even after controlling for cortical thickness. Moreover, for participants with MCI, gray matter NDI-but not cortical thickness-was lower in temporal, parietal, and posterior cingulate regions. Taken together, our results highlight the utility of NODDI metrics in detecting cortical microstructural degeneration that occurs prior to measurable macrostructural changes and overt clinical dementia., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Structural biology and functional studies are a powerful combination to elucidate fundamental knowledge about the cystic fibrosis transmembrane conductance regulator (CFTR). Here, we discuss the latest findings, including how clinically-approved drugs restore function to mutant CFTR, leading to better clinical outcomes for people with cystic fibrosis (CF). Despite the prospect of regulatory approval of a CFTR-targeting therapy for most CF mutations, strenuous efforts are still needed to fully comprehend CFTR structure-and-function for the development of better drugs to enable people with CF to live full and active lives., Competing Interests: Declaration of Competing Interest DNS is the recipient of a Vertex Innovation Award, IS-G is the recipient of two Vertex Innovation Awards and a member of the scientific boards of Eloxx, PTC Therapeutics and Vertex Therapeutics. T-CH has an ongoing service agreement with Abbvie and a sponsored research grant from Nanova., (Copyright © 2019. Published by Elsevier B.V.)

Pullulanases are well-known debranching enzymes hydrolyzing α-1,6-glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis-involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the 'evolutionary coupling saturation mutagenesis' (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity., (© 2019 Federation of European Biochemical Societies.)

Metalloproteins comprise over one-third of proteins, with approximately half of all enzymes requiring metal to function. Accurate identification of these metal atoms and their environment is a prerequisite to understanding biological mechanism. Using ion beam analysis through particle induced X-ray emission (PIXE), we have quantitatively identified the metal atoms in 30 previously structurally characterized proteins using minimal sample volume and a high-throughput approach. Over half of these metals had been misidentified in the deposited structural models. Some of the PIXE detected metals not seen in the models were explainable as artifacts from promiscuous crystallization reagents. For others, using the correct metal improved the structural models. For multinuclear sites, anomalous diffraction signals enabled the positioning of the correct metals to reveal previously obscured biological information. PIXE is insensitive to the chemical environment, but coupled with experimental diffraction data deposited alongside the structural model it enables validation and potential remediation of metalloprotein models, improving structural and, more importantly, mechanistic knowledge.

The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Members of an important group of industrial enzymes, Rhizopus lipases, exhibit valuable hydrolytic features that underlie their biological functions. Particularly important is their N-terminal polypeptide segment (NTPS), which is required for secretion and proper folding but is removed in the process of enzyme maturation. A second common feature of this class of lipases is the α-helical "lid", which regulates the accessibility of the substrate to the enzyme active site. Some Rhizopus lipases also exhibit "interfacial activation" by micelle and/or aggregate surfaces. While it has long been recognized that the NTPS is critical for function, its dynamic features have frustrated efforts to characterize its structure by X-ray crystallography. Here, we combine nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the structure and dynamics of Rhizopus chinensis lipase (RCL) with its 27-residue NTPS prosequence (r27RCL). Both r27RCL and the truncated mature form of RCL (mRCL) exhibit biphasic interfacial activation kinetics with p -nitrophenyl butyrate ( p NPB). r27RCL exhibits a substrate binding affinity significantly lower than that of mRCL due to stabilization of the closed lid conformation by the NTPS. In contrast to previous predictions, the NTPS does not enhance lipase activity by increasing surface hydrophobicity but rather inhibits activity by forming conserved interactions with both the closed lid and the core protein structure. Single-site mutations and kinetic studies were used to confirm that the NTPS serves as internal competitive inhibitor and to develop a model of the associated process of interfacial activation. These structure-function studies provide the basis for engineering RCL lipases with enhanced catalytic activities.

Recombinant proteins are essential for biotechnology. Here we review some of the key points for improving the production of heterologous proteins, and what can be the future of the field., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: An association of acetaminophen use and asthma was observed in the International Study of Asthma and Allergies in Childhood study. However there are no clear mechanisms to explain an association between acetaminophen use and immunologic pathology. In acidic conditions like those in the stomach and inflamed airway, tyrosine residues are nitrated by nitrous and peroxynitrous acids. The resulting nitrotyrosine is structurally similar to 2,4-dinitrophenol and 2,4-dinitrochlorobenzene, known haptens that enhance immune responses by covalently binding proteins. Nitrated acetaminophen shares similar molecular structure., Objective: We hypothesized the acetaminophen phenol ring undergoes nitration under acidic conditions, producing 3-nitro-acetaminophen which augments allergic responses by acting as a hapten for environmental allergens., Methods: 3-nitro-acetaminophen was formed from acetaminophen in the presence of acidified nitrite, purified by high performance liquid chromatography, and assayed by gas-chromatography mass spectrometry. Purified 3-nitro-acetaminophen was reacted with Dermatophagoides pteronyssinus (Der p1) and analyzed by mass spectrometry to identify the modification site. Human peripheral blood mononuclear cells proliferation response was measured in response to 3-nitro-acetaminophen and to 3-nitro-acetaminophen-modified Der p1., Results: Acetaminophen was modified by nitrous acid forming 3-nitro-acetaminophen over a range of different acidic conditions consistent with airway inflammation and stomach acidity. The Der p1 protein-hapten adduct creation was confirmed by liquid chromatography-mass spectrometry proteomics modifying cysteine 132. Peripheral blood mononuclear cells exposed to 3-nitro-acetaminophen-modified Der p1 had increased proliferation and cytokine production compared to acetaminophen and Der p1 alone (n = 7; p < 0.05)., Conclusion: These data suggests 3-nitro-acetaminophen formation and reaction with Der p1 provides a mechanism by which stomach acid or infection-induced low airway pH in patients could enhance the allergic response to proteins such as Der p1.

Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using 13 C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, 13 C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2 H, 13 C, 15 N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology., Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2)., (© 2017 Federation of European Biochemical Societies.)

This paper presents experimental results of both quasi-static compression and low-velocity impact behavior for tri-axial bio-composite structural panels using a spherical load head. Panels were made having different core and face configurations. The results showed that panels made having either carbon fiber fabric composite faces or a foam-filled core had significantly improved impact and compressive performance over panels without either. Different localized impact responses were observed based on the location of the compression or impact relative to the tri-axial structural core; the core with a smaller structural element had better impact performance. Furthermore, during the early contact phase for both quasi-static compression and low-velocity impact tests, the panels with the same configuration had similar load-displacement responses. The experimental results show basic compression data could be used for the future design and optimization of tri-axial bio-composite structural panels for potential impact applications.

Synonymous variations in protein-coding sequences alter protein expression dynamics, which has important implications for cellular physiology and evolutionary fitness, but disentangling the underlying molecular mechanisms remains challenging., (Copyright © 2017 Elsevier Inc. All rights reserved.)

DUF1537 is a novel family of kinases identified by comparative genomic approaches. The family is widespread and found in all sequenced plant genomes and 16% of sequenced bacterial genomes. DUF1537 is not a monofunctional family and contains subgroups that can be separated by phylogenetic and genome neighborhood context analyses. A subset of the DUF1537 proteins is strongly associated by physical clustering and gene fusion with the PdxA2 family, demonstrated here to be a functional paralog of the 4-phosphohydroxy-l-threonine dehydrogenase enzyme (PdxA), a central enzyme in the synthesis of pyridoxal-5'-phosphate (PLP) in proteobacteria. Some members of this DUF1537 subgroup phosphorylate l-4-hydroxythreonine (4HT) into 4-phosphohydroxy-l-threonine (4PHT), the substrate of PdxA, in vitro and in vivo. This provides an alternative route to PLP from the toxic antimetabolite 4HT that can be directly generated from the toxic intermediate glycolaldehyde. Although the kinetic and physical clustering data indicate that these functions in PLP synthesis are not the main roles of the DUF1537-PdxA2 enzymes, genetic and physiological data suggest these side activities function has been maintained in diverse sets of organisms.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development., (© 2015 The Protein Society.)

Design of complex alpha-beta protein topologies poses a challenge because of the large number of alternative packing arrangements. A similar challenge presumably limited the emergence of large and complex protein topologies in evolution. Here, we demonstrate that protein topologies with six and seven-stranded beta sheets can be designed by insertion of one de novo designed beta sheet containing protein into another such that the two beta sheets are merged to form a single extended sheet, followed by amino acid sequence optimization at the newly formed strand-strand, strand-helix, and helix-helix interfaces. Crystal structures of two such designs closely match the computational design models. Searches for similar structures in the SCOP protein domain database yield only weak matches with different beta sheet connectivities. A similar beta sheet fusion mechanism may have contributed to the emergence of complex beta sheets during natural protein evolution.

Exhaled breath condensate (EBC) pH serves as a surrogate marker of airway lining fluid (ALF) pH and can be used to evaluate airway acidification (AA). AA is known to be present in acute respiratory distress syndrome (ARDS) and can be evaluated via continuous EBC pH measurement during mechanical ventilation. Lung recruitment maneuvers (LRMs) are utilized in the treatment of ARDS, however, their impact on EBC pH has never been explored. Here we described the acute effects of two commonly used LRMs on EBC pH. In a prospective, non-randomized, serial exposure study, 10 intubated pediatric subjects with acute respiratory distress syndrome sequentially underwent: a period of baseline ventilation, sustained inflation (SI) maneuver of 40 cm H2O for 40 s, open lung ventilation, staircase recruitment strategy (SRS) (which involves a systematic ramping of plateau pressures in 5 cm H2O increments, starting at 30 cm H2O), and PEEP titration. Maximum lung recruitment during the SRS is defined as a PaO2 + PaCO2 of  >400 mmHg. Following lung recruitment, PEEP titration was conducted from 20 cm H2O in 2 cm H2O decrements until a PaO2 + PaCO2 was  <380 and then increased by 2 cm H2O. EBC pH, arterial blood gases, lung mechanics, hemodynamics, and function residual capacity were obtained following each phase of the LRM and observational period. Seven out of 10 patients were able to reach maximum lung recruitment. Baseline EBC pH (6.38   ±   0.37) did not correlate with disease severity defined by PaO2/FiO2 ratio or oxygenation index (OI). Average EBC pH differed between phases and decreased after LRM (p = 0.001). EBC pH is affected by LRMs. EBC acidification following LRMs may represent a washout effect of opening acidic lung units and ventilating them or acute AA resulting from LRM.

A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The TenA protein family occurs in prokaryotes, plants and fungi; it has two subfamilies, one (TenA&#95;C) having an active-site cysteine, the other (TenA&#95;E) not. TenA&#95;C proteins participate in thiamin salvage by hydrolysing the thiamin breakdown product amino-HMP (4-amino-5-aminomethyl-2-methylpyrimidine) to HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine); the function of TenA&#95;E proteins is unknown. Comparative analysis of prokaryote and plant genomes predicted that (i) TenA&#95;E has a salvage role similar to, but not identical with, that of TenA&#95;C and (ii) that TenA&#95;E and TenA&#95;C also have non-salvage roles since they occur in organisms that cannot make thiamin. Recombinant Arabidopsis and maize TenA&#95;E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, hydrolysed the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a line with a null mutation in the HMP biosynthesis gene ThiC prevented its rescue by amino-HMP. Ablating At3g16990 in the wild-type increased sensitivity to paraquat-induced oxidative stress; HMP overcame this increased sensitivity. Furthermore, the expression of TenA&#95;E and ThiC genes in Arabidopsis and maize was inversely correlated. These results indicate that TenA&#95;E proteins mediate amidohydrolase and aminohydrolase steps in the salvage of thiamin breakdown products. As such products can be toxic, TenA&#95;E proteins may also pre-empt toxicity.

Queuosine (Q) is a modification found at the wobble position of tRNAs with GUN anticodons. Although Q is present in most eukaryotes and bacteria, only bacteria can synthesize Q de novo. Eukaryotes acquire queuine (q), the free base of Q, from diet and/or microflora, making q an important but under-recognized micronutrient for plants, animals, and fungi. Eukaryotic type tRNA-guanine transglycosylases (eTGTs) are composed of a catalytic subunit (QTRT1) and a homologous accessory subunit (QTRTD1) forming a complex that catalyzes q insertion into target tRNAs. Phylogenetic analysis of eTGT subunits revealed a patchy distribution pattern in which gene losses occurred independently in different clades. Searches for genes co-distributing with eTGT family members identified DUF2419 as a potential Q salvage protein family. This prediction was experimentally validated in Schizosaccharomyces pombe by confirming that Q was present by analyzing tRNA(Asp) with anticodon GUC purified from wild-type cells and by showing that Q was absent from strains carrying deletions in the QTRT1 or DUF2419 encoding genes. DUF2419 proteins occur in most Eukarya with a few possible cases of horizontal gene transfer to bacteria. The universality of the DUF2419 function was confirmed by complementing the S. pombe mutant with the Zea mays (maize), human, and Sphaerobacter thermophilus homologues. The enzymatic function of this family is yet to be determined, but structural similarity with DNA glycosidases suggests a ribonucleoside hydrolase activity.

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be time-consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and to the insertion and correct placement of sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient, CDcoeff, appears to be robust in this application, and coupled with hierarchical clustering and the overlay of crystallization outcome, reveals information of biological relevance. While tested with a single example the potential applications related to crystallography appear promising and the distance coefficient, clustering, and hierarchal visualization of results undoubtedly have applications in wider fields.

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification., (© 2014 The Protein Society.)

A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents.

Manure from beef cattle feedyards is a valuable source of nutrients and assists with maintaining soil quality. However, humification and decomposition processes occurring during feedyard manure's on-farm life cycle influence the forms, concentrations, and availability of carbon (C) and nutrients such as nitrogen (N) and phosphorus (P). Improved understanding of manure organic matter (OM) chemistry will provide better estimates of potential fertilizer value of manure from different feedyard sources (e.g., manure accumulated in pens, stockpiled manure after pen scraping) and in settling basin and retention pond sediments. This will also assist with identifying factors related to nutrient loss and environmental degradation via volatilization of ammonia and nitrous oxide and nitrate leaching. We used Fourier-transform infrared (FTIR) and ultraviolet-visible (UV-vis) spectroscopies to characterize structural and functional properties of OM and water-extractable OM (WEOM) from different sources (surface manure, manure pack, settling basin, retention pond) on a typical commercial beef feedyard in the Texas Panhandle. Results showed that as beef manure completes its on-farm life cycle, concentrations of dissolved organic C and N decrease up to 98 and 95%, respectively. The UV-vis analysis of WEOM indicated large differences in molecular weight, lignin content, and proportion of humified OM between manures from different sources. The FTIR spectra of OM and WEOM indicate preferential decomposition of fats, lipids, and proteins over aromatic polysaccharides such as lignin. Further work is warranted to evaluate how application of feedyard manure from different sources influences soil metabolic functioning and fertility., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein energy-dependent translational throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistently with this inference, EttA-deleted cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a new mechanism sensitive to cellular energy status.

Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, energy-dependent translational throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA-binding site. Using single-molecule fluorescence resonance energy transfer, we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, in which the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches., (© 2013.)

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.

While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding., (© 2013.)

NanoRNase (Nrn) specifically degrades nucleoside 3',5'-bisphosphate and the very short RNA, nanoRNA, during the final step of mRNA degradation. The crystal structure of Nrn in complex with a reaction product GMP was determined. The overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. Two Mn²⁺ ions are coordinated by conserved residues in the DHH motif of the N-terminal domain. GMP binds near the DHHA1 motif region in the C-terminal domain. Our structure enables us to predict the substrate-bound form of Nrn as well as other DHH/DHHA1 phosphoesterase family proteins., (Copyright © 2013 Federation of European Biochemical Societies. All rights reserved.)

Background: Airway acidification plays a role in disorders of the pulmonary tract. We hypothesized that the inhalation of alkalinized glycine buffer would measurably alkalinize the airways without compromising lung function or causing adverse events. We evaluated the safety of an inhaled alkaline glycine buffer in both healthy subjects and in subjects with stable obstructive airway disease., Methods: This work includes 2 open-label safety studies. The healthy controls were part of a phase 1 safety study of multiple inhalations of low-dose alkaline glycine buffer; nebulized saline was used as a comparator in 8 of the healthy controls. Subsequently, a phase 2 study in subjects with stable obstructive airway disease was completed using a single nebulized higher-dose strategy of the alkaline inhalation. We studied 20 non-smoking adults (10 healthy controls and 10 subjects with obstructive airway disease), both at baseline and after inhalation of alkaline buffer. We used spirometry and vital signs as markers of clinical safety. We used changes in fraction of exhaled nitric oxide (NO) and exhaled breath condensate (EBC) pH as surrogate markers of airway pH modification., Results: Alkaline glycine inhalation was tolerated by all subjects in both studies, with no adverse effects on spirometric parameters or vital signs. Airway alkalinization was confirmed by a median increase in EBC pH of 0.235 pH units (IQR 0.56-0.03, P = .03) in subjects after inhalation of the higher-dose alkaline buffer (2.5 mL of 100 mmol/L glycine)., Conclusions: Alkalinization of airway lining fluid is accomplished with inhalation of alkaline glycine buffer and causes no adverse effects on pulmonary function or vital signs.

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.

The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.

SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ~100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ~250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes.

Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.