N-terminal Structure Research Articles

Switchable reinforced streptavidin.

The complex of the small molecule biotin and the homotetrameric protein streptavidin is key to a broad range of biotechnological applications. Therefore, the behavior of this extraordinarily high-affinity interaction under mechanical force is intensively studied by single-molecule force spectroscopy. Recently, steered molecular dynamics simulations have identified a low force pathway for the dissociation of biotin from streptavidin, which involves partial unfolding of the N-terminal β-sheet structure of monovalent streptavidin's functional subunit. Based on these results, we now introduced two mutations (T18C,A33C) in the functional subunit of monovalent streptavidin to establish a switchable connection (disulfide bridge) between the first two β-strands to prevent this unfolding. In atomic force microscopy-based single-molecule force spectroscopy experiments, we observed unbinding forces of about 350 pN (at a force-loading rate of 10 nN s-1) for pulling a single biotin out of an N-terminally anchored monovalent streptavidin binding pocket - about 1.5-fold higher compared with what has been reported for N-terminal force loading of native monovalent streptavidin. Upon addition of a reducing agent, the unbinding forces dropped back to 200 pN, as the disulfide bridge was destroyed. Switching from reducing to oxidizing buffer conditions, the inverse effect was observed. Our work illustrates how the mechanics of a receptor-ligand system can be tuned by engineering the receptor protein far off the ligand-binding pocket.

Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Removal of N-terminal tail changes the thermostability of the low-temperature-active exo-inulinase InuAGN25

ABSTRACT Exo-inulinases are members of the glycoside hydrolase family 32 and function by hydrolyzing inulin into fructose with yields up to 90–95%. The N-terminal tail contributes to enzyme thermotolerance, which plays an important role in enzyme applications. However, the role of N-terminal amino acid residues in the thermal performance and structural properties of exo-inulinases remains to be elucidated. In this study, three and six residues of the N-terminus starting from Gln23 of the exo-inulinase InuAGN25 were deleted and expressed in Escherichia coli. After digestion with human rhinovirus 3 C protease to remove the N-terminal amino acid fusion sequence that may affect the thermolability of enzymes, wild-type RfsMInuAGN25 and its mutants RfsMutNGln23Δ3 and RfsMutNGln23Δ6 were produced. Compared with RfsMInuAGN25, thermostability of RfsMutNGln23Δ3 was enhanced while that of RfsMutNGln23Δ6 was slightly reduced. Compared with the N-terminal structures of RfsMInuAGN25 and RfsMutNGln23Δ6, RfsMutNGln23Δ3 had a higher content of (1) the helix structure, (2) salt bridges (three of which were organized in a network), (3) cation–π interactions (one of which anchored the N-terminal tail). These structural properties may account for the improved thermostability of RfsMutNGln23Δ3. The study provides a better understanding of the N-terminus–function relationships that are useful for rational design of thermostability of exo-inulinases.

Local conformations affect the histidine tag-Ni<sup>2+</sup> binding affinity of BinA and BinB proteins

Binary toxin (Bin) is one of the bio-larvicidal toxin produced by <em>Lysinibacillus sphaericus</em>. Two component proteins, BinA and BinB toxins are required simultaneously to exert its larvicidal activity. The binary toxin has been proposed to act initially on the susceptible cell membrane. Here, the cell membrane binding of the binary toxin was imitated via specific histidine (His)-nickel ion (Ni²⁺) chelating. The N-terminal His-conjugated binary toxins (His-Bin) were attached onto the Ni²⁺-lipid bilayer surface besides its facilitating of purification process. The N-terminal conjugation of histidine tag did not interfere with the folding structure of both toxins. Subsequently, the attachment of binary toxins on the lipid membrane was successful with Ni²⁺-phosphatidylcholine (POPC)/phosphatidylethanolamine (POPE) bilayers (a model membrane that mimics the mosquito cell membrane) but not for Ni²⁺-phosphatidylcholine bilayer. However, His-BinA formed unstable attachment with Ni²⁺-POPC/POPE bilayers since it could be removed by buffer rinsing. In contrast, His-BinB required imidazole solution to detach from Ni²⁺-POPC/POPE. Particularly, His-BinB had higher binding affinity to Ni²⁺-ion than His-BinA. The lipid membrane attachment led to the initial finding that although BinA and BinB toxins share high homology structures, their capability for Ni²⁺ chelation was different. The local N-terminal structure of binary toxin seems to interfere the His-Ni²⁺ chelating of His-BinA.

Biophysical studies on the antimicrobial activity of linearized esculentin 2EM

Linearized esculentin 2 EM (E2EM-lin) from the frog, Glandirana emeljanovi was highly active against Gram-positive bacteria (minimum lethal concentration ≤ 5.0 μM) and strongly α-helical in the presence of lipid mimics of their membranes (>55.0%). The N-terminal α-helical structure adopted by E2EM-lin showed the potential to form a membrane interactive, tilted peptide with an hydrophobicity gradient over residues 9 to 23. E2EM-lin inserted strongly into lipid mimics of membranes from Gram-positive bacteria (maximal surface pressure changes ≥5.5 mN m−1), inducing increased rigidity (Cs−1 ↑), thermodynamic instability (ΔGmix < 0 → ΔGmix > 0) and high levels of lysis (>50.0%). These effects appeared to be driven by the high anionic lipid content of membranes from Gram-positive bacteria; namely phosphatidylglycerol (PG) and cardiolipin (CL) species. The high levels of α-helicity (60.0%), interaction (maximal surface pressure change = 6.7 mN m−1) and lysis (66.0%) shown by E2EM-lin with PG species was a major driver in the ability of the peptide to lyse and kill Gram-positive bacteria. E2EM-lin also showed high levels of α-helicity (62.0%) with CL species but only low levels of interaction (maximal surface pressure change = 2.9 mN m−1) and lysis (21.0%) with the lipid. These combined data suggest that E2EM-lin has a specificity for killing Gram-positive bacteria that involves the formation of tilted structure and appears to be primarily driven by PG-mediated membranolysis. These structure/function relationships are used to help explain the pore forming process proposed to describe the membranolytic, antibacterial action of E2EM-lin.

Genome-Wide Analysis of the YABBY Transcription Factor Family in Pineapple and Functional Identification of AcYABBY4 Involvement in Salt Stress.

The plant-specific transcription factor gene family, YABBY, belongs to the subfamily of zinc finger protein superfamily and plays an essential regulatory role in lateral organ development. In this study, nine YABBY genes were identified in the pineapple genome. Seven of them were located on seven different chromosomes and the remaining two were located on scaffold 1235. Through protein structure prediction and protein multiple sequence alignment, we found that AcYABBY3, AcYABBY5 and AcYABBY7 lack a C2 structure in their N-terminal C2C2 zinc finger protein structure. Analysis of the cis-acting element indicated that all the seven pineapple YABBY genes contain multiple MYB and MYC elements. Further, the expression patterns analysis using the RNA-seq data of different pineapple tissues indicated that different AcYABBYs are preferentially expressed in various tissues. RT-qPCR showed that the expression of AcYABBY2, AcYABBY3, AcYABBY6 and AcYABBY7 were highly sensitive to abiotic stresses. Subcellular localization in pineapple protoplasts, tobacco leaves and Arabidopsis roots showed that all the seven pineapple YABBY proteins were nucleus localized. Overexpression of AcYABBY4 in Arabidopsis resulted in short root under NaCl treatment, indicating a negative regulatory role of AcYABBY4 in plant resistance to salt stress. This study provides valuable information for the classification of pineapple AcYABBY genes and established a basis for further research on the functions of AcYABBY proteins in plant development and environmental stress response.

Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia.

Transcriptome sequencing data (6.5 Gb) of the salivary glands of the haematophagous leech Hirudo nipponia was obtained by using the BGIseq-500 platform. After identification and analysis, one transcript (Unigene5370) was annotated to hirudin HV3 from Hirudo medicinalis with an e-value of 1e-29 and was named hirudin-HN. This transcript was a new thrombin inhibitor gene belonging to the proteinase inhibitor I14 (hirudin) family. Hirudin-HN, with a 270-bp cDNA, encodes an 89-aa protein containing a 20-aa signal peptide. The mature hirudin-HN protein contains the typical structural characteristics of hirudin, e.g., three conserved disulfide bonds and the PKP and DFxxIP motifs. Proteins (Hir and M-Hir) were obtained via prokaryotic expression, and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance (SPR) interaction analysis, respectively. The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity of Hir and M-Hir. The abundances of Hirudin-HN mRNA and protein were higher in the salivary glands of starving animals than in those of feeding or fed leeches. These results provided a foundation for further study on the structure-function relationship of hirudin-HN with thrombin.

Abstract 1775: Targeting RSK4 prevents both chemoresistance and metastasis in lung cancer

Abstract Lung cancer is the commonest cause of cancer death worldwide with a five-year survival rate of less than five percent for metastatic tumors. Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer cases of which adenocarcinoma prevails. Patients almost invariably develop metastatic drug-resistant disease and this is responsible for our failure to provide curative therapy. Hence, a better understanding of the mechanisms underlying these biological processes is urgently required to improve clinical outcome. The 90-kDa ribosomal S6 kinases (RSKs) are downstream effectors of the RAS/MAPK cascade. RSKs are highly conserved serine/threonine protein kinases implicated in diverse cellular processes, including cell survival, proliferation, migration and invasion. Four isoforms exist in humans (RSK1-4) and are uniquely characterized by the presence of two non-identical N- and C-terminal kinase domains. RSK isoforms are 73-80% identical at protein level and this has been thought to suggest overlapping functions. However, through functional genomic kinome screens, we show that RSK4, contrary to RSK1, promotes both drug resistance and metastasis in lung cancer. This kinase is overexpressed in the majority (57%) of NSCLC biopsies and this correlates with poor overall survival in lung adenocarcinoma patients. Genetic silencing of RSK4 sensitizes lung cancer cells to chemotherapy and prevents their migration and invasiveness in vitro and in vivo. RSK4 downregulation decreases the anti-apoptotic proteins Bcl2 and cIAP1/2 which correlates with increased apoptotic signalling, whilst it also induces mesenchymal-epithelial transition (MET) through inhibition of NFκB activity. A small-molecule inhibitor screen identified several floxacins, including trovafloxacin, as potent allosteric inhibitors of RSK4 activation. Trovafloxacin reproduced all biological and molecular effects of RSK4 silencing in vitro and in vivo, and is predicted to bind a novel allosteric site revealed by our RSK4 N-terminal kinase domain crystal structure and mathematical Markov Transient Analysis. Taken together, our data demonstrate that RSK4 represents a promising novel therapeutic target in lung cancer. Citation Format: Stelios Chrysostomou, Rajat Roy, Filippo Prischi, Katie Chapman, Uwais Mufti, Francesco Mauri, Guido Bellezza, Joel Abrahams, Silvia Ottaviani, Leandro Castellano, Georgios Giamas, David Hrouda, Mathias Winkler, David Klug, Sophia Yaliraki, Mauricio Barahona, Yulan Wang, Maruf Ali, Michael Seckl, Olivier Pardo. Targeting RSK4 prevents both chemoresistance and metastasis in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1775.

The Cytoplasm-Entry Domain of Antibacterial CdiA Is a Dynamic α-Helical Bundle with Disulfide-Dependent Structural Features

Many Gram‐negative bacterial species use contact-dependent growth inhibition (CDI) systems to compete with neighboring cells. CDI+ strains express cell-surface CdiA effector proteins, which carry a toxic C-terminal region (CdiA-CT) that is cleaved from the effector upon transfer into the periplasm of target bacteria. The released CdiA-CT consists of two domains. The C-terminal domain is typically a nuclease that inhibits cell growth, and the N-terminal “cytoplasm-entry” domain mediates toxin translocation into the target-cell cytosol. Here, we use NMR and circular dichroism spectroscopic approaches to probe the structure, stability, and dynamics of the cytoplasm-entry domain from Escherichia coli STEC_MHI813. Chemical shift analysis reveals that the CdiA-CTMHI813 entry domain is composed of a C-terminal helical bundle and a dynamic N-terminal region containing two disulfide linkages. Disruption of the disulfides by mutagenesis or chemical reduction destabilizes secondary structure over the N-terminus, but has no effect on the C-terminal helices. Although critical for N-terminal structure, the disulfides have only modest effects on global thermodynamic stability, and the entry domain exhibits characteristics of a molten globule. We find that the disulfides form in vivo as the entry domain dwells in the periplasm of inhibitor cells prior to target-cell recognition. CdiA-CTMHI813 variants lacking either disulfide still kill target bacteria, but disruption of both bonds abrogates growth inhibition activity. We propose that the entry domain's dynamic structural features are critical for function. In its molten globule-like state, the domain resists degradation after delivery, yet remains pliable enough to unfold for membrane translocation.

Alternative σI/anti-σI factors represent a unique form of bacterial σ/anti-σ complex.

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of σI and anti-σI factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-σ factors that have N-terminal helical structures, RsgIN has a β-barrel structure. Unlike other anti-σ factors that bind both σ2 and σ4 domains of the σ factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter –35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different σI/anti-σI pairs. We suggest that the σI/anti-σI factors represent a distinctive mode of σ/anti-σ complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate σI/anti-σI system.

All-atom structure ensembles of islet amyloid polypeptides determined by enhanced sampling and experiment data restraints.

Exploring the accurate structure ensembles are critical to understand the functions of intrinsically disordered proteins (IDPs). As a well-known IDP, islet amyloid polypeptide (IAPP) plays important roles in the development of human type II diabetes (T2D). The toxicity of human IAPP (hIAPP) is induced by the amyloidosis of the peptide, however, its aggregation mechanism remains ambiguous. The characterization of structure ensemble of hIAPP, as well as the differences between hIAPP and its non-amyloidogenic homologous such as rat IAPP (rIAPP), would greatly help to illuminate the amyloidosis mechanism of IAPP. In this study, the atomic structure ensembles of hIAPP and rIAPP were characterized by all-atom molecular dynamics (MD) simulations combined with enhanced sampling technology and experiment data restraints. The obtained structure ensembles were firstly compared with those determined by the conventional MD (cMD) and enhanced sampling without experiment data restraints. The results showed that the enhanced sampling and experiment data restraints would improve the simulation accuracy. The transient N-terminal α-helix structures were adopted by the sub-states of both hIAPP and rIAPP, however, the C-terminal helical structures were only present on hIAPP. The hydrophobic residues in the amyloid-core region of hIAPP are exposed to the solvent. The structure ensemble differences between hIAPP and rIAPP revealed in this work provide potential explain to the amyloidogenic mechanism and would be helpful for the design of drugs to combat T2D.

Molecular dynamics simulation on the effect of transition metal binding to the N-terminal fragment of amyloid-β

We report molecular dynamics simulations of three possible adducts of Fe(II) to the N-terminal 1–16 fragments of the amyloid-β peptide, along with analogous simulations of Cu(II) and Zn(II) adducts. We find that multiple simulations from different starting points reach pseudo-equilibration within 100–300 ns, leading to over 900 ns of equilibrated trajectory data for each system. The specifics of the coordination modes for Fe(II) have only a weak effect on peptide secondary and tertiary structures, and we therefore compare one of these with analogous models of Cu(II) and Zn(II) complexes. All share broadly similar structural features, with mixture of coil, turn and bend in the N-terminal region and helical structure for residues 11–16. Within this overall pattern, subtle effects due to changes in metal are evident: Fe(II) complexes are more compact and are more likely to occupy bridge and ribbon regions of Ramachandran maps, while Cu(II) coordination leads to greater occupancy of the poly-proline region. Analysis of representative clusters in terms of molecular mechanics energy and atoms-in-molecules properties indicates similarity of four-coordinate Cu and Zn complexes, compared to five-coordinate Fe complex that exhibits lower stability and weaker metal–ligand bonding.Communicated by Ramaswamy H. Sarma

Distinct APE1 Activities Affect the Regulation of VEGF Transcription Under Hypoxic Conditions

Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF), a crucial factor in tumor angiogenesis, has been reported to be transcriptionally regulated by hypoxia-inducible factor-1 (HIF-1). An 8-oxo-G or apurinic/apyrimidinic (AP) site, which is frequently associated with DNA damage, has been identified in the promoter region of VEGF. However, the detailed molecular mechanisms by which AP sites regulate VEGF gene transcription are largely unknown. The dual functional protein apurinic/apyrimidinic endonuclease 1 (APE1) is both the key enzyme in DNA base excision repair and the redox factor shown to regulate HIF-1 DNA-binding activity. In the present study, we tested the involvement of both the AP endonuclease and redox activity of APE1 in regulating HIF-1 DNA binding and VEGF transcription in HUVECs. By employing two APE1 activity-specific inhibitors and AP-site-containing reporter constructs, we confirmed that both activities of APE1 were involved in regulating VEGF expression under hypoxic conditions. Furthermore, we found that the interaction between APE1 and its downstream repair enzyme, DNA polymerase β, was compromised when the N-terminal structure of APE1 was distorted under oxidative conditions. Our data suggest that the DNA repair and redox activity of APE1 can play a collaborative role in regulating the transcriptional initiation of the AP-site-containing promoter.

Dynamics of the Centromeric Histone CENH3 Structure in Rye-Wheat Amphidiploids (Secalotriticum).

The centromeres perform integral control of the cell division process and proper distribution of chromosomes into daughter cells. The correct course of this process is often disrupted in case of remote hybridization, which is a stress factor. The combination of parental genomes of different species in a hybrid cell leads to a “genomic shock” followed by loss of genes, changes in gene expression, deletions, inversions, and translocations of chromosome regions. The created rye-wheat allopolyploid hybrids, which were collectively called secalotriticum, represent a new interesting model for studying the effect of remote hybridization on the centromere and its components. The main feature of an active centromere is the presence of a specific histone H3 modification in the centromeric nucleosomes, which is referred to as CENH3 in plants. In this paper the results of cytogenetic analysis of the secalotriticum hybrid karyotypes and the comparison of the CENH3 N-terminal domain structure of parent and hybrid forms are presented. It is shown that the karyotypes of the created secalotriticum forms are stable balanced hexaploids not containing minichromosomes with deleted arms, in full or in part. A high level of homology between rye and wheat enables to express both parental forms of CENH3 gene in the hybrid genomes of secalotriticum cultivars. The CENH3 structure in hybrids in each crossing combination has some specific features. The percentage of polymorphisms at several amino acid positions is much higher in one of the secalotriticum hybrids, STr VD, than in parental forms, whereas the other hybrid, STr VM, inherits a high level of amino acid substitutions at the position 25 from the maternal parent.

Dissociation of [b5 – H][rad]+ ions composed of one α-methyltryptophan and four alanine residues: The effect of the α-methyl group

Collision-induced dissociation (CID) of hexapeptide radical cations consisting of four alanine residues, one α-methyltryptophan and a glycine residue at the C-terminus results predominantly in bond cleavages around the sterically crowded α-methyltryptophan residue, but all generated [b5 – H]+ ions in moderate abundance. Unlike in the dissociations of the [b5 – H]+ ions of the analogous [A4Woxa]+ ions, where regardless of the initial location of the unmodified tryptophan residue the CID spectra were identical, the α-methyltryptophan-containing [b5 – H]+ ions all gave different CID spectra, indicating that there was no evidence of isomerization. Losses of imine-amides and the neutral of mass 141 Da provide evidence that the radical centre is located at the α-carbon of the N-terminal residue. This N-terminal captodative structure inhibits macrocyclization, thereby preventing isomerization and leading to different dissociation products. Density functional theory (DFT) calculations on [AAAWα-MeAoxa]+, the ion that gave the largest variety of dissociation products, established that the captodative structure at the N-terminus is at the global minimum, 27 kJ mol−1 lower in energy than the π-radical cation. Calculations also showed that losses of the dialanine radical from the captodative structure and the side chain of α-methyltryptophan from the π-radical are the two lowest-energy dissociation pathways, consistent with the onsets observed in the experimental energy-resolved CID curves. The overall conclusion is that [b5 – H]+ ions that contain an unmodified tryptophan have the radical centre located on the α-carbon of the tryptophan residue, and this leaves the N-terminal amino group free to initiate macrocyclization leading to isomerization prior to dissociation. When the α-carbon is blocked by a methyl group, as in α-methyltryptophan, then the captodative structure at the N-terminal residue is favoured and this prevents macrocyclization and isomerization.

Effect of Disulfide Bond Incorporation on the Structure and Activity of Endostatin Peptide.

The structure and function of a 27-a.a. fragment of the N-terminal sequence of human endostatin (ES-Zn) were compared to those of the mutant peptide (ES-SSZn) obtained by adding Cys-Pro-Ala to the endostatin N-terminus and substituting Asn16 for Cys ensuring formation of a disulfide bond. Structural comparison of ES-Zn and ES-SSZn by far-UV circular dichroism (CD), intrinsic fluorescence, and molecular dynamics simulation methods revealed significant structural perturbations in ES-SSZn, such as elimination of the β-sheet conformer, modification of the N-terminal loop structure, and reorganization of dynamic properties of the entire peptide backbone. ES-SSZn was approximately 2 and 3 times less efficient than ES-Zn and the full-length human endostatin, respectively, in the induction of caspase-3-dependent apoptosis in human umbilical vein endothelial cells (HUVECs) in vitro (p < 0.05). In contrast, treatment of metastatic 4T1 breast tumors in mice with ES-Zn and ES-SSZn (5 mg/kg body weight daily) for 14 days resulted in similar regression of tumor size, comparable downregulation of angiogenesis (CD31 and CD34) and cell proliferation (Ki67), and therefore, the same extent of apoptosis induction (TUNEL, p53, and Bcl-2) for both peptides (as compared to the untreated controls). Western blot analysis of HUVEC and 4T1 tumor lysates revealed the same levels of suppression of key signaling mediators Akt and ERK1/2 by ES-Zn and ES-SSZn. Contrary to the earlier studies, our results showed that the function of the 1-27 endostatin fragment is independent of its overall structure. Stabilization of the N-terminal loop structure by the disulfide bond incorporation causes relief from structural deviations.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

Elucidation of the N‐terminal Structure and Characteristics of Perilipin 5

The United States and other nations are experiencing an epidemic of lipid‐related diseases. Understanding the etiologies of these diseases is an increasingly imperative objective. Finding the function and mechanisms of the molecules involved in these lipid‐related diseases could show new and enhanced treatments for those suffering from these conditions. The perilipins, a family of five conserved lipid droplet proteins, are a chief subject of study in this field. However, an abundance of structural data is absent and would facilitate major strides in clarifying perilipin function. There is three‐dimensional structural data on the carboxyl terminus of one member in the perilipin family, perilipin 3, but not for other family members. The current study is aimed to characterize and determine the structure of the N‐terminal domain of perilipin 5. Perilipin 5 is of interest because of its implications in numerous of the metabolic conditions observed in the population. The N‐terminus is the concentration of the project because the C‐terminus of the protein has a high sequence homology with that of perilipin 3. Therefore, the C‐terminus of perilipin 5 is predicted to take on a similar structure to the C‐terminus of perilipin 3. A cDNA fragment coding for the N‐terminal region of perilipin 5 was PCR amplified from the full‐length Mus musculus perilipin 5 gene. It was then transformed into Escherichia Coli to produce the protein fragment of interest. Isolation of the protein fragment is undergoing via nickel affinity chromatography. Theoretical secondary and tertiary structures of the N‐terminal fragment of perilipin 5 have been obtained using the programs Phyre2, SCRATCH, GOR4, CFSSP, and Jpred. Computer models predict the sequence's secondary structure to contain numerous alpha helices with increasing frequency near the back half of the fragment. In conclusion, determination of the N‐terminal structure of perilipin 5 will further enable research into the functions of the perilipin family.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fibrillation of N-Terminal Prion Protein Fragment in Presence of Zinc Ions

Abnormal (misfolded) form of human prion protein displays high propensity towards self-association which may result in formation of insoluble fibrillar deposits in the brain, associated with the development of fatal neurodegenerative disorders, transmissible spongiform encephalopathies (TSE) [1]. The formation of such deposits can be driven by many intra- and extracellular factors. One of them can be disrupted homeostasis of metal ions. As reported previously, Zn ions bonded to N-terminal fragment regulate the interactions between N- and C-terminals [2]. Here we present structural analysis of the fragment of PrP N-terminal domain in the presence of Zn ions. Our experiments demonstrate that Zn-saturation leads to formation of N-terminal domain fibrillar structures. The fibrils were analyzed by atomic force microscopy (AFM), electron microscopy (EM), X-ray fiber diffraction and X-ray absorption spectroscopy (XAS). AFM and EM images indicated, that in the presence of zinc, PrP peptide forms long fibrillar structures. The presence of two characteristic reflections at 1.12 nm and 0.36 nm in the X-ray diffraction pattern confirmed the fibrillar nature of the studied PrP peptide. These reflections corresponded to characteristic distances in the fibrillar structure of the studied PrP peptide. Zn XAS K-edge 1s->4p transition in the edge region indicates, that Zn occupies a mononuclear site and is coordinated by light atoms (N, O). Fourier transform of χ(k) signal indicates the presence of imidazole groups in the nearest Zn shell. Our findings may shed new light on the role of zinc in TSE disorders. This work was supported by the funds from the National Science Centre (Poland) granted on the basis of decision no. 2014/15/B/ST4/04839.[1] Kovacs, G. G., & Budka, H. (2008). The American Journal of Pathology, 172(3), 555-565. [2] Spevacek, A.R., et al. (2013) Structure, 21, 236-246.