Expanding and reprogramming the genetic code of cells and animals.

Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.

Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response.

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.

Selenomethionine as an expressible handle for bioconjugations.

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, is selectively labile to glutathione, and embodies a broadly tunable cleavage profile. Specifically, a 4-bromomethylphenylacetyl (BrMePAA) linker has been applied for efficient conjugation of complex organic molecules to SeM-containing proteins. This expansion of the bioconjugation toolkit has broad potential in the development of chemically enhanced proteins.

Structure-function characterization of the mono- and diheme forms of MhuD, a noncanonical heme oxygenase from Mycobacterium tuberculosis.

MhuD is a noncanonical heme oxygenase (HO) from Mycobacterium tuberculosis (Mtb) that catalyzes unique heme degradation chemistry distinct from canonical HOs, generating mycobilin products without releasing carbon monoxide. Its crucial role in the Mtb heme uptake pathway has identified MhuD as an auspicious drug target. MhuD is capable of binding either one or two hemes within a single active site, but only the monoheme form was previously reported to be enzymatically active. Here we employed resonance Raman (rR) spectroscopy to examine several factors proposed to impact the reactivity of mono- and diheme MhuD, including heme ruffling, heme pocket hydrophobicity, and amino acid-heme interactions. We determined that the distal heme in the diheme MhuD active site has negligible effects on both the planarity of the His-coordinated heme macrocycle and the strength of the Fe-NHis linkage relative to the monoheme form. Our rR studies using isotopically labeled hemes unveiled unexpected biomolecular dynamics for the process of heme binding that converts MhuD from mono- to diheme form, where the second incoming heme replaces the first as the His75-coordinated heme. Ferrous CO-ligated diheme MhuD was found to exhibit multiple Fe-C-O conformers, one of which contains catalytically predisposed H-bonding interactions with the distal Asn7 residue identical to those in the monoheme form, implying that it is also enzymatically active. This was substantiated by activity assays and MS product analysis that confirmed the diheme form also degrades heme to mycobilins, redefining MhuD's functional paradigm and further expanding our understanding of its role in Mtb physiology.

Active site serine-193 modulates activity of human aromatic amino acid decarboxylase.

Aromatic amino acid decarboxylase is a pyridoxal 5'-phosphate-dependent enzyme responsible for the synthesis of the neurotransmitters, dopamine and serotonin. Here, by a combination of bioinformatic predictions and analyses, phosphorylation assays, spectroscopic investigations and activity measurements, we determined that Ser-193, a conserved residue located at the active site, can be phosphorylated, increasing catalytic efficiency. In order to determine the molecular basis for this functional improvement, we determined the structural and kinetic properties of the site-directed variants S193A, S193D and S193E. While S193A retains 27% of the catalytic efficiency of wild-type, the two acidic side chain variants are impaired in catalysis with efficiencies of about 0.15% with respect to the wild-type. Thus, even if located at the active site, Ser-193 is not essential for enzyme activity. We advance the idea that this residue is fundamental for the correct architecture of the active site in terms of network of interactions triggering catalysis. This role has been compared with the properties of the Ser-194 of the highly homologous enzyme histidine decarboxylase whose catalytic loop is visible in the spatial structure, allowing us to propose the validation for the effect of the phosphorylation. The effect could be interesting for AADC deficiency, a rare monogenic disease, whose broad clinical phenotype could be also related to post translational AADC modifications.

Mechanism of dimerization and structural features of human LI-cadherin.

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion-free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin-dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.

Polyglutamylation: biology and analysis.

Polyglutamylation is a posttranslational modification (PTM) that adds several glutamates on glutamate residues in the form of conjugated peptide chains by a family of enzymes known as polyglutamylases. Polyglutamylation is well documented in microtubules. Polyglutamylated microtubules consist of different α- and ß-tubulin subunits with varied number of added glutamate residues. Kinetic control and catalytic rates of tubulin modification by polyglutamylases influence the polyglutamylation pattern of functional microtubules. The recent studies uncovered catalytic mechanisms of the glutamylation enzymes family, particularly tubulin tyrosine ligase-like (TTLL). Variable length polyglutamylation of primary sequence glutamyl residues have been mapped with a multitude of protein chemistry and proteomics approaches. Although polyglutamylation was initially considered a tubulin-specific modification, the recent studies have uncovered a calmodulin-dependent glutamylase, SidJ. Nano-electrospray ionization (ESI) proteomic approaches have identified quantifiable polyglutamylated sites in specific substrates. Indeed, conjugated glutamylated peptides were used in nano-liquid chromatography gradient delivery due to their relative hydrophobicity for their tandem mass spectrometry (MS/MS) characterization. The recent polyglutamylation characterization has revealed three major sites: E445 in α-tubulin, E435 in ß-tubulin, and E860 in SdeA. In this review, we have summarized the progress made using proteomic approaches for large-scale detection of polyglutamylated peptides, including biology and analysis.

Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

Exploring cellular biochemistry with nanobodies.

Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

Templated folding of intrinsically disordered proteins.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity.

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB-substrate complexes. Furthermore, we also demonstrated that residues 163-168 located on the ß6/ß7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

Characterizing posttranslational modifications in prokaryotic metabolism using a multiscale workflow.

Understanding the complex interactions of protein posttranslational modifications (PTMs) represents a major challenge in metabolic engineering, synthetic biology, and the biomedical sciences. Here, we present a workflow that integrates multiplex automated genome editing (MAGE), genome-scale metabolic modeling, and atomistic molecular dynamics to study the effects of PTMs on metabolic enzymes and microbial fitness. This workflow incorporates complementary approaches across scientific disciplines; provides molecular insight into how PTMs influence cellular fitness during nutrient shifts; and demonstrates how mechanistic details of PTMs can be explored at different biological scales. As a proof of concept, we present a global analysis of PTMs on enzymes in the metabolic network of Escherichia coli Based on our workflow results, we conduct a more detailed, mechanistic analysis of the PTMs in three proteins: enolase, serine hydroxymethyltransferase, and transaldolase. Application of this workflow identified the roles of specific PTMs in observed experimental phenomena and demonstrated how individual PTMs regulate enzymes, pathways, and, ultimately, cell phenotypes.

Covalent Probes for Aggregated Protein Imaging via Michael Addition.

Covalent chemical reactions to modify aggregated proteins are rare. Here, we reported covalent Michael addition can generally occur upon protein aggregation. Such reactivity was initially discovered by a bioinspired fluorescent color-switch probe mimicking the photo-conversion mechanism of Kaede fluorescent protein. This probe was dark with folded proteins but turned on red fluorescence (620 nm) when it non-covalently bound to misfolded proteins. Supported by the biochemical and mass spectrometry results, the probe chemoselectively reacted with the reactive cysteines of aggregated proteins via covalent Michael addition and gradually switched to green fluorescence (515 nm) upon protein aggregation. Exploiting this Michael addition chemistry in the malachite green dye derivatives demonstrated its general applicability and chemical tunability, resulting in different fluorescence color-switch responses. Our work may offer a new avenue to explore other chemical reactions upon protein aggregation and design covalent probes for imaging, chemical proteomics, and therapeutic purposes.

Allosteric disulfides: Sophisticated molecular structures enabling flexible protein regulation.

Protein disulfide bonds link pairs of cysteine residues in polypeptide chains. Many of these bonds serve a purely structural or energetic role, but a growing subset of cleavable disulfide bonds has been shown to control the function of the mature protein in which they reside. These allosteric disulfides and the factors that cleave these bonds are being identified across biological systems and life forms and have been shown to control hemostasis, the immune response, and viral infection in mammals. The discovery of these functional disulfides and a rationale for their facile nature has been aided by the emergence of a conformational signature for allosteric bonds. This post-translational modification mostly occurs extracellularly, making these chemical events prime drug targets. Indeed, a membrane-impermeable inhibitor of one of the cleaving factors is currently being trialed as an antithrombotic agent in cancer patients. Allosteric disulfides are firmly established as a sophisticated means by which a protein's shape and function can be altered; however, the full scope of this biological regulation will not be realized without new tools and techniques to study this regulation and innovative ways of targeting it.

Targetable Tetrazine-Based Dynamic Nuclear Polarization Agents for Biological Systems.

Dynamic nuclear polarization (DNP) has shown great promise as a tool to enhance the nuclear magnetic resonance signals of proteins in the cellular environment. As sensitivity increases, the ability to select and efficiently polarize a specific macromolecule over the cellular background has become desirable. Herein, we address this need and present a tetrazine-based DNP agent that can be targeted selectively to proteins containing the unnatural amino acid (UAA) norbornene-lysine. This UAA can be introduced efficiently into the cellular milieu by genetic means. Our approach is bio-orthogonal and easily adaptable to any protein of interest. We illustrate the scope of our methodology and investigate the DNP transfer mechanisms in several biological systems. Our results shed light on the complex polarization-transfer pathways in targeted DNP and ultimately pave the way to selective DNP-enhanced NMR spectroscopy in both bacterial and mammalian cells.

New variants of AADC deficiency expand the knowledge of enzymatic phenotypes.

AADC deficiency is a rare genetic disease caused by mutations in the gene of aromatic amino acid decarboxylase, the pyridoxal 5'-phosphate dependent enzyme responsible for the synthesis of dopamine and serotonin. Here, following a biochemical approach together with an in silico bioinformatic analysis, we present a structural and functional characterization of 13 new variants of AADC. The amino acid substitutions are spread over the entire protein from the N-terminal (V60A), to its loop1 (H70Y and F77L), to the large domain (G96R) and its various motifs, i.e. loop2 (A110E), or a core ß-barrel either on the surface (P210L, F251S and E283A) or in a more hydrophobic milieu (L222P, F237S and W267R) or loop3 (L353P), and to the C-terminal domain (R453C). Results show that the ß-barrel variants exhibit a low solubility and those belonging to the surface tend to aggregate in their apo form, leading to the identification of a new enzymatic phenotype for AADC deficiency. Moreover, five variants of residues belonging to the large interface of AADC (V60A, G96R, A110E, L353P and R453C) are characterized by a decreased catalytic efficiency. The remaining ones (H70Y and F77L) present features typical of apo-to-holo impaired transition. Thus, defects in catalysis or in the acquirement of the correct holo structure are due not only to specific local domain effects but also to long-range effects at either the protein surface or the subunit interface. Altogether, the new characterized enzymatic phenotypes represent a further step in the elucidation of the molecular basis for the disease.

Water is an active matrix of life for cell and molecular biology.

Szent-Gyorgi called water the "matrix of life" and claimed that there was no life without it. This statement is true, as far as we know, on our planet, but it is not clear whether it must hold throughout the cosmos. To evaluate that question requires a close consideration of the many varied and subtle roles that water plays in living cells-a consideration that must be free of both an assumed essentialism that gives water an almost mystical life-giving agency and a traditional tendency to see it as a merely passive solvent. Water is a participant in the "life of the cell," and here I describe some of the features of that active agency. Water's value for molecular biology comes from both the structural and dynamic characteristics of its status as a complex, structured liquid as well as its nature as a polar, protic, and amphoteric reagent. Any discussion of water as life's matrix must, however, begin with an acknowledgment that our understanding of it as both a liquid and a solvent is still incomplete.

The radical SAM protein HemW is a heme chaperone.

Radical S-adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S-C(5') bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes. For example, heme chaperones insert heme into target proteins but have been studied only for the formation of cytochrome c-type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer but not for stable heme binding. A bacterial two-hybrid system screen identified bacterioferritins and the heme-containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins.

Hydrogen sulfide bypasses the rate-limiting oxygen activation of heme oxygenase.

Discovery of unidentified protein functions is of biological importance because it often provides new paradigms for many research areas. Mammalian heme oxygenase (HO) enzyme catalyzes the O2-dependent degradation of heme into carbon monoxide (CO), iron, and biliverdin through numerous reaction intermediates. Here, we report that H2S, a gaseous signaling molecule, is part of a novel reaction pathway that drastically alters HO's products, reaction mechanism, and catalytic properties. Our prediction of this interplay is based on the unique reactivity of H2S with one of the HO intermediates. We found that in the presence of H2S, HO produces new linear tetrapyrroles, which we identified as isomers of sulfur-containing biliverdin (SBV), and that only H2S, but not GSH, cysteine, and polysulfides, induces SBV formation. As BV is converted to bilirubin (BR), SBV is enzymatically reduced to sulfur-containing bilirubin (SBR), which shares similar properties such as antioxidative effects with normal BR. SBR was detected in culture media of mouse macrophages, confirming the existence of this H2S-induced reaction in mammalian cells. H2S reacted specifically with a ferric verdoheme intermediate of HO, and verdoheme cleavage proceeded through an O2-independent hydrolysis-like mechanism. This change in activation mode diminished O2 dependence of the overall HO activity, circumventing the rate-limiting O2 activation of HO. We propose that H2S could largely affect O2 sensing by mammalian HO, which is supposed to relay hypoxic signals by decreasing CO output to regulate cellular functions. Moreover, the novel H2S-induced reaction identified here helps sustain HO's heme-degrading and antioxidant-generating capacity under highly hypoxic conditions.