Mass spectrometric studies of peptides. III. AUTOMATED determination of amino acid sequences
Mass spectrometric determination of amino acid sequences in peptides
AMINO ACID↗
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The synthesis of some peptides related to the active site of enzymes.
Synthesis of peptides related to active site of esteratic enzymes
PEPTIDE↗
Synthesis of biologically pertinent peptides under possible primordial conditions.
Synthesis of biologically pertinent peptides under possible primordial conditions
BIOGENESIS↗
Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution
The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.
Non-NASA Center↗
Peptide nucleic acids rather than RNA may have been the first genetic molecule
Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.
NASA Discipline Exobiology↗
Histidine‐Mediated Ion Specific Effects Enable Salt Tolerance of a Pore‐Forming Marine Antimicrobial Peptide
Abstract Antimicrobial peptides (AMPs) preferentially permeate prokaryotic membranes via electrostatic binding and membrane remodeling. Such action is drastically suppressed by high salt due to increased electrostatic screening, thus it is puzzling how marine AMPs can possibly work. We examine as a model system, piscidin‐1, a histidine‐rich marine AMP, and show that ion‐histidine interactions play unanticipated roles in membrane remodeling at high salt: Histidines can simultaneously hydrogen‐bond to a phosphate and coordinate with an alkali metal ion to neutralize phosphate charge, thereby facilitating multidentate bonds to lipid headgroups in order to generate saddle‐splay curvature, a prerequisite to pore formation. A comparison among Na + , K + , and Cs + indicates that histidine‐mediated salt tolerance is ion specific. We conclude that histidine plays a unique role in enabling protein/peptide‐membrane interactions that occur in marine or other high‐salt environment.
Xian, Wujing↗
Programmed Supramolecular Assemblies Using Orthogonal Pairs of Heterodimeric Coiled Coil Peptides
Despite recent progress, it remains challenging to program biomacromolecules to assemble into discrete nanostructures with pre-determined sizes and topologies. We report here a novel strategy to address this challenge. By using two orthogonal pairs of heterodimeric coiled coils as the building blocks, we constructed six discrete supramolecular assemblies, each composed of a prescribed number of coiled coil components. Within these assemblies, different coiled coils were connected via end-to-side covalent linkages strategically pre-installed between the non-complementary pairs. The overall topological features of two highly complex assemblies, a “barbell” and a “quadrilateral” form, were characterized experimentally and were in good agreement to the designs. This work expands the design paradigms for peptide-based discrete supramolecular assemblies and will provide a route for de novo fabrication of functional protein materials.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Prebiotic Membranes and Micelles Do Not Inhibit Peptide Formation During Dehydration
Abstract Cycles of dehydration and rehydration could have enabled formation of peptides and RNA in otherwise unfavorable conditions on the early Earth. Development of the first protocells would have hinged upon colocalization of these biopolymers with fatty acid membranes. Using atomic force microscopy, we find that a prebiotic fatty acid (decanoic acid) forms stacks of membranes after dehydration. Using LC‐MS‐MS (liquid chromatography‐tandem mass spectrometry) with isotope internal standards, we measure the rate of formation of serine dipeptides. We find that dipeptides form during dehydration at moderate temperatures (55 °C) at least as fast in the presence of decanoic acid membranes as in the absence of membranes. Our results are consistent with the hypothesis that protocells could have formed within evaporating environments on the early Earth.
Cohen, Zachary R.↗
The Role of Asparagine as a Gatekeeper Residue in the Selective Binding of Rare Earth Elements by Lanthanide‐Binding Peptides
Abstract Lanthanide‐binding tag (LBT) peptides selectively complex lanthanide cations (Ln 3+ ) in their binding pockets and are promising for lanthanide separation. However, designing LBTs that selectively target specific Ln 3+ cations remains a challenge due to limited molecular‐level understanding and control of interactions within the lanthanide‐binding pocket. In this study, we reveal that the N5 asparagine residue acts as a gatekeeper in the binding pocket, resulting in a 100‐fold selectivity for smaller Lu 3+ over larger La 3+ cations. Nuclear magnetic resonance spectroscopy and molecular dynamics simulations show that the N5 residue weakly binds to the larger La 3+ cation, permitting H 2 O molecules inside the pocket. For the smaller Lu 3+ cations, the N5 residue forms an inter‐arm hydrogen bond with the E14 glutamic acid residue, locking the Lu 3+ cation in the pocket and preventing H 2 O infiltration. Mutating the N5 asparagine to a D5 aspartic acid prevents such a hydrogen bond, eliminating the gatekeeping mechanism and precipitously reducing selectivity. The resulting binding affinity to Ln 3+ cations is non‐monotonic but generally increases with cation size. These results suggest a molecular design paradigm: the reduced affinity for larger lanthanides is due to open pocket conformations, while the selectivity of smaller Ln 3+ cations over larger ones is due to the gatekeeping hydrogen bond.
Chemistry↗
Persistence of Methionine Side Chain Mobility at Low Temperatures in a Nine‐Residue Low Complexity Peptide, as Probed by 2 H Solid‐State NMR
Abstract Methionine side chains are flexible entities which play important roles in defining hydrophobic interfaces. We utilize deuterium static solid‐state NMR to assess rotameric inter‐conversions and other dynamic modes of the methionine in the context of a nine‐residue random‐coil peptide (RC9) with the low‐complexity sequence GGKGMGFGL. The measurements in the temperature range of 313 to 161 K demonstrate that the rotameric interconversions in the hydrated solid powder state persist to temperatures below 200 K. Removal of solvation significantly reduces the rate of the rotameric motions. We employed 2 H NMR line shape analysis, longitudinal and rotation frame relaxation, and chemical exchange saturation transfer methods and found that the combination of multiple techniques creates a significantly more refined model in comparison with a single technique. Further, we compare the most essential features of the dynamics in RC9 to two different methionine‐containing systems, characterized previously. Namely, the M35 of hydrated amyloid‐β 1–40 in the three‐fold symmetric polymorph as well as Fluorenylmethyloxycarbonyl (FMOC)‐methionine amino acid with the bulky hydrophobic group. The comparison suggests that the driving force for the enhanced methionine side chain mobility in RC9 is the thermodynamic factor stemming from distributions of rotameric populations, rather than the increase in the rate constant.
Vugmeyster, Liliya↗
Precision Loading and Delivery of Molecular Cargo by Size-Controlled Coacervation of Gold Nanoparticles Functionalized with Elastin-like Peptides
Thermoresponsive elastin-like peptides (ELPs) have been extensively investigated in biotechnology and medicine, but little attention has been paid to the process by which coacervation causes ELP-decorated particles to aggregate. Here, using gold nanoparticles (AuNPs) functionalized with a cysteine-terminated 96-repeat of the VPGVG sequence (V96-Cys), we show that the size of the clusters that reversibly form above the ELP transition temperature can be finely controlled in the 250 to 930 nm range by specifying the concentration of free V96-Cys in solution and using AuNPs of different sizes. We further find that the localized surface plasmon resonance peak of the embedded AuNPs progressively red-shifts with cluster size, likely due to an increase in particle–particle contacts. We exploit this fine control over size to homogeneously load precise amounts of the dye Nile Red and the antibiotic Tetracycline into clusters of different hydrodynamic diameters and deliver cargos near-quantitatively by deconstructing the aggregates below the ELP transition temperature. Beyond establishing a key role for free ELPs in the agglomeration of ELP-functionalized particles, our results provide a path for the thermally controlled delivery of precise quantities of molecular cargo. This capability might prove useful in combination photothermal therapies and theranostic applications, and to trigger spatially and temporally uniform responses from biological, electronic, or optical systems.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Selective One-Dimensional 13 C– 13 C Spin-Diffusion Solid-State Nuclear Magnetic Resonance Methods to Probe Spatial Arrangements in Biopolymers Including Plant Cell Walls, Peptides, and Spider Silk
Two-dimensional (2D) and 3D through-space 13 C– 13 C homonuclear spin-diffusion techniques are powerful solid-state nuclear magnetic resonance (NMR) tools for extracting structural information from 13 C-enriched biomolecules, but necessarily long acquisition times restrict their applications. In this work, we explore the broad utility and underutilized power of a chemical shift-selective one-dimensional (1D) version of a 2D 13 C– 13 C spin-diffusion solid-state NMR technique. The method, which is called 1D dipolar-assisted rotational resonance (DARR) difference, is applied to a variety of biomaterials including lignocellulosic plant cell walls, microcrystalline peptide fMLF, and black widow dragline spider silk. 1D 13 C– 13 C spin-diffusion methods described here apply in select cases in which the 1D 13 C solid-state NMR spectrum displays chemical shift-resolved moieties. Furthermore, this is analogous to the selective 1D nuclear Overhauser effect spectroscopy (NOESY) experiment utilized in liquid-state NMR as a faster (1D instead of 2D) and often less ambiguous (direct sampling of the time domain data, coupled with increased signal averaging) alternative to 2D NOESY. Selective 1D 13 C– 13 C spin-diffusion methods are more time-efficient than their 2D counterparts such as proton-driven spin diffusion (PDSD) and dipolar-assisted rotational resonance. The additional time gained enables measurements of 13 C– 13 C spin-diffusion buildup curves and extraction of spin-diffusion time constants TSD, yielding detailed structural information. Specifically, selective 1D DARR difference buildup curves applied to 13 C-enriched hybrid poplar woody stems confirm strong spatial interaction between lignin and acetylated xylan polymers within poplar plant secondary cell walls, and an interpolymer distance of ~0.45–0.5 nm was estimated. Additionally, Tyr/Gly long-range correlations were observed on isotopically enriched black widow spider dragline silks.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Peptide Selenocysteine Substitutions Reveal Direct Substrate–Enzyme Interactions at Auxiliary Clusters in Radical S -Adenosyl- L -methionine Maturases
Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe–4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C–H to initiate catalysis, followed by formation of a C–S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se–C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Designing Peptide Fossils That Model the Evolution of the Bacterial Ferredoxin Fold
Electron transfer coupled to redox chemistry is at the heart of metabolism. The proteins responsible for moving electrons (protein electron carriers) must have emerged at the origin of life. The small iron–sulfur-binding bacterial ferredoxins were likely among these first proteins. Embedded within the ferredoxin sequence and structure is a symmetry that points to an ancient gene duplication event. Little is understood about the nature of ferredoxins prior to this duplication event or what environmental factors may have driven the selection for more complex forms. The deep-time molecular history of ferredoxins goes back billions of years and cannot be reconstructed by phylogenetic analyses based on amino acid sequences. Here, we use structure-guided protein design to model a fossil half-ferredoxin stage in the evolution of this fold, the semidoxins, and their symmetric full-length counterparts, the symdoxins. Semidoxin designs homodimerize, exhibiting structural, thermodynamic, and electrochemical behaviors in most cases identical to cognate symdoxins. However, the semi- and symdoxin fossil stages behave differently when incorporated into an in vivo electron transfer complementation assay. Both can support bacterial growth dependent on protein expression. Growth rates of bacteria expressing the semidoxins are much more sensitive to oxygen than those of bacteria expressing symdoxins. Motivated by the in vivo functionality of designed semidoxins, we identified putative naturally occurring semidoxins in extant anaerobic microorganisms. This is consistent with the observed in vivo oxygen sensitivity of the semidoxin designs. One natural semidoxin is shown to be folded and redox active. However, it exists as a mixture of monomers and dimers, suggesting a potential connection between semidoxins and even simpler single iron–sulfur cluster-binding peptides.
59 BASIC BIOLOGICAL SCIENCES↗
Integrating genomics and metabolomics for scalable non-ribosomal peptide discovery
Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.
59 BASIC BIOLOGICAL SCIENCES↗
Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis
Abstract Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.
59 BASIC BIOLOGICAL SCIENCES↗
Molecular basis of differential HLA class I-restricted T cell recognition of a highly networked HIV peptide
Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design.
60 APPLIED LIFE SCIENCES↗
Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site
The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.
60 APPLIED LIFE SCIENCES↗