Current Protein & Peptide Science[JOURNAL]
Sun
168 papers
Molecular design and genetic optimization of antimicrobial peptides containing unnatural amino acids against antibiotic-resistant bacterial infections.
He Y, He X
Biopolymers · 2016 Sep · PMID 27258330 · Publisher ↗
Antimicrobial peptides (AMPs) have been the focus of intense research towards the finding of a viable alternative to current small-molecule antibiotics, owing to their commonly observed and naturally occurring resistance...
Antimicrobial peptides (AMPs) have been the focus of intense research towards the finding of a viable alternative to current small-molecule antibiotics, owing to their commonly observed and naturally occurring resistance against pathogens. However, natural peptides have many problems such as low bioavailability and high allergenicity that largely limit the clinical applications of AMPs. In the present study, an integrative protocol that combined chemoinformatics modeling, molecular dynamics simulations, and in vitro susceptibility test was described to design AMPs containing unnatural amino acids (AMP-UAAs). To fulfill this, a large panel of synthetic AMPs with determined activity was collected and used to perform quantitative structure-activity relationship (QSAR) modeling. The obtained QSAR predictors were then employed to direct genetic algorithm (GA)-based optimization of AMP-UAA population, to which a number of commercially available, structurally diverse unnatural amino acids were introduced during the optimization process. Subsequently, several designed AMP-UAAs were confirmed to have high antibacterial potency against two antibiotic-resistant strains, i.e. multidrug-resistant Pseudomonas aeruginosa (MDRPA) and methicillin-resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentration (MIC) < 10 μg/ml. Structural dynamics characterizations revealed that the most potent AMP-UAA peptide is an amphipathic helix that can spontaneously embed into an artificial lipid bilayer and exhibits a strong destructuring tendency associated with the embedding process. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 746-756, 2016.
Review: Mechanochemistry of the kinesin-1 ATPase.
Cross RA
Biopolymers · 2016 Aug · PMID 27120111 · Full text
Kinesins are P-loop NTPases that can do mechanical work. Like small G-proteins, to which they are related, kinesins execute a program of active site conformational changes that cleaves the terminal phosphate from an NTP...
Kinesins are P-loop NTPases that can do mechanical work. Like small G-proteins, to which they are related, kinesins execute a program of active site conformational changes that cleaves the terminal phosphate from an NTP substrate. But unlike small G-proteins, kinesins can amplify and harness these conformational changes in order to exert force. In this short review I summarize current ideas about how the kinesin active site works and outline how the active site chemistry is coupled to the larger-scale structural cycle of the kinesin motor domain. Focusing largely on kinesin-1, the best-studied kinesin, I discuss how the active site switch machinery of kinesin cycles between three distinct states, how docking of the neck linker stabilizes two of these states, and how tension-sensitive and position-sensitive neck linker docking may modulate both the hydrolysis step of ATP turnover and the trapping of product ADP in the active site. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 476-482, 2016.
Review: Structure and mechanism of the dynein motor ATPase.
Schmidt H, Carter AP
Biopolymers · 2016 Aug · PMID 27062277 · Full text
Dyneins are multiprotein complexes that move cargo along microtubules in the minus end direction. The largest individual component of the dynein complex is the heavy chain. Its C-terminal 3500 amino-acid residues form th...
Dyneins are multiprotein complexes that move cargo along microtubules in the minus end direction. The largest individual component of the dynein complex is the heavy chain. Its C-terminal 3500 amino-acid residues form the motor domain, which hydrolyses ATP in its ring of AAA+ (ATPases associated with diverse cellular activities) domains to generate the force for movement. The production of force is synchronized with cycles of microtubule binding and release, another important prerequisite for efficient motility along the microtubule. Although the large scale conformational changes that lead to force production and microtubule affinity regulation are well established, it has been largely enigmatic how ATP-hydrolysis in the AAA+ ring causes these rearrangements. The past five years have seen a surge of high resolution information on the dynein motor domain that finally allowed unprecedented insights into this important open question. This review, part of the "ATP and GTP hydrolysis in Biology" special issue, will summarize our current understanding of the dynein motor mechanism with a special emphasis on the recently obtained crystal and EM structures. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 557-567, 2016.
Review: The ATPase mechanism of myosin and actomyosin.
Geeves MA
Biopolymers · 2016 Aug · PMID 27061920 · Publisher ↗
Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicat...
Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 483-491, 2016.
Oxidation of the N-terminal domain of the wheat metallothionein Ec -1 leads to the formation of three distinct disulfide bridges.
Tarasava K, Chesnov S, Freisinger E
Biopolymers · 2016 May · PMID 27061576 · Publisher ↗
Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal...
Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal-thiolate clusters. Depending on intracellular conditions such as redox potential or metal ion concentrations, MTs can occur in various states ranging from the fully metal-loaded holo- to the metal-free apo-form. The Cys thiolate groups in the apo-form can be either reduced or be involved in disulfide bridges. Although oxidation-mediated Zn(II) release might be a possible mechanism for the regulation of Zn(II) availability by MTs, no concise information regarding the associated pathways and the structure of oxidized apo-MT forms is available. Using the well-studied Zn2 γ-Ec -1 domain of the wheat Zn6 Ec -1 MT we attempt here to answer several question regarding the structure and biophysical properties of oxidized MT forms, such as: (1) does disulfide bond formation increase the stability against proteolysis, (2) is the overall peptide backbone fold similar for the holo- and the oxidized apo-MT form, and (3) are disulfide bridges specifically or randomly formed? Our investigations show that oxidation leads to three distinct disulfide bridges independently of the applied oxidation conditions and of the initial species used for oxidation, that is, the apo- or the holo-form. In addition, the oxidized apo-form is as stable against proteolysis as Zn2 γ-Ec -1, rendering the currently assumed degradation of oxidized MTs unlikely and suggesting a role of the oxidation process for the extension of protein lifetime in absence of sufficient amounts of metal ions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 295-308, 2016.
Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading.
Bailey ME, Jiang N, Dima RI … +1 more , Ross JL
Biopolymers · 2016 Aug · PMID 27037673 · Publisher ↗
Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced...
Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule-severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP-enzymes. Here we review current knowledge about GTP-driven microtubule dynamics and how that couples to ATP-driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 547-556, 2016.
Invited review: Small GTPases and their GAPs.
Mishra AK, Lambright DG
Biopolymers · 2016 Aug · PMID 26972107 · Full text
Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bou...
Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bound inactive states under control of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which accelerate slow intrinsic rates of activation by nucleotide exchange and deactivation by GTP hydrolysis, respectively. Here we review developments leading to current understanding of intrinsic and GAP catalyzed GTP hydrolytic reactions in small GTPases from structural, molecular and chemical mechanistic perspectives. Despite the apparent simplicity of the GTPase cycle, the structural bases underlying the hallmark hydrolytic reaction and catalytic acceleration by GAPs are considerably more diverse than originally anticipated. Even the most fundamental aspects of the reaction mechanism have been challenging to decipher. Through a combination of experimental and in silico approaches, the outlines of a consensus view have begun to emerge for the best studied paradigms. Nevertheless, recent observations indicate that there is still much to be learned. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 431-448, 2016.
Salt-resistant short antimicrobial peptides.
Mohanram H, Bhattacharjya S
Biopolymers · 2016 May · PMID 26849911 · Publisher ↗
Antimicrobial peptides (AMPs) are promising leads for the development of antibiotics against drug resistant bacterial pathogens. However, in vivo applications of AMPs remain obscure due to salt and serum mediated inactiv...
Antimicrobial peptides (AMPs) are promising leads for the development of antibiotics against drug resistant bacterial pathogens. However, in vivo applications of AMPs remain obscure due to salt and serum mediated inactivation. The high cost of chemical synthesis of AMPs also impedes potential clinical application. Consequently, short AMPs resistant toward salt and serum inactivation are desirable for the development of peptide antibiotics. In this work, we designed a 12-residue amphipathic helical peptide RR12 (R-R-L-I-R-L-I-L-R-L-L-R-amide) and two Trp containing analogs of RR12 namely RR12Wpolar (R-R-L-I-W-L-I-L-R-L-L-R-amide), and RR12Whydro (R-R-L-I-R-L-W-L-R-L-L-R-amide). Designed peptides demonstrated potent antibacterial activity; MIC ranging from 2 to 8 μM, in the presence of sodium chloride (150 mM and 300 mM). Antibacterial activity of these peptides was also detected in the presence of human serum. Designed peptides, in particular RR12 and RR12Whydro, were only poorly hemolytic. As a mode of action; these peptides demonstrated efficient permeabilization of bacterial cell membrane and lysis of cell structure. We further investigated interactions of the designed peptides with lipopolysaccharide (LPS), the major component of the outer membrane permeability barrier of Gram-negative bacteria. Designed peptides adopted helical conformations in complex with LPS. Binding of peptides with LPS has yielded dissociation the aggregated structures of LPS. Collectively, these designed peptides hold ability to be developed for salt-resistant antimicrobial compounds. Most importantly, current work provides insights for designing salt-resistant antimicrobial peptides. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 345-356, 2016.
Optimization of adiponectin-derived peptides for inhibition of cancer cell growth and signaling.
Otvos L, Kovalszky I, Olah J … +7 more , Coroniti R, Knappe D, Nollmann FI, Hoffmann R, Wade JD, Lovas S, Surmacz E
Biopolymers · 2015 May · PMID 25683126 · Publisher ↗
Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we i...
Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10-mer ADP355) and an antagonist (8-mer ADP400) to modulate various adiponectin receptor-mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14-mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5-10-fold improved agonistic activity were the replacement of turn-forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native β-sheet structure. All peptides retained good stability in human serum exhibiting half-lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure-activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin-induced signals can result in cytostatic and anti-oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo-aca (placed to the N-terminus) and ADP355 (at the C-terminus). The in vitro anti-tumor activity and intracellular signaling of the chimera were dominated by the more active Allo-aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist.
Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.
Qian WJ, Park JE, Lim D … +7 more , Lai CC, Kelley JA, Park SY, Lee KW, Yaffe MB, Lee KS, Burke TR
Biopolymers · 2014 Nov · PMID 25283071 · Full text
Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-contain...
Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.
The C-terminal calcium-sensitive disordered motifs regulate isoform-specific polymerization characteristics of calsequestrin.
Bal NC, Jena N, Chakravarty H … +7 more , Kumar A, Chi M, Balaraju T, Rawale SV, Rawale JS, Sharon A, Periasamy M
Biopolymers · 2015 Jan · PMID 25091206 · Full text
Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Inter...
Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C-terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn-motif and DEXn-motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C-terminal motifs possess Ca(2+)-sensitivity and affect protein function. Sequence analysis shows that both the Dn- and DEXn-motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca(2+)-mediated folding suggesting that these disordered motifs are Ca(2+)-sensitivity. We generated chimeric proteins by swapping the C-terminal portions between CASQ1 and CASQ2. Our studies show that the C-terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C-terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca(2+)-sensitivity IDRs located at the back-to-back dimer interface influence isoform-specific Ca(2+)-dependent polymerization properties of CASQ.
Creation of catalytically active particles from enzymes crosslinked with a natural bifunctional agent--homocysteine thiolactone.
Stroylova YY, Semenyuk PI, Asriyantz RA … +3 more , Gaillard C, Haertlé T, Muronetz VI
Biopolymers · 2014 Sep · PMID 24912753 · Publisher ↗
The current study describes an approach to creation of catalytically active particles with increased stability from enzymes by N-homocysteinylation, a naturally presented protein modification. Enzymatic activities and pr...
The current study describes an approach to creation of catalytically active particles with increased stability from enzymes by N-homocysteinylation, a naturally presented protein modification. Enzymatic activities and properties of two globular tetrameric enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were studied before and after N-homocysteinylation. Modification of these proteins concerns the accessible lysine residues and introduces an average of 2-2,5 homocysteine residues per protein monomer. Formation of a range of aggregates was observed for both enzymes, which assemble via formation of intermolecular noncovalent bonds and by disulfide bonds. It was demonstrated that both studied enzymes retain their catalytic activities on modification and the subsequent formation of oligomeric forms. At low concentrations of homocysteine thiolactone, modification of GAPDH leads not only to prevention of spontaneous inactivation but also increases thermal stability of this enzyme on heating to 80°C. A moderate reduction of the activity of GAPDH observed in case of its crosslinking with 50-fold excess of homocysteine thiolactone per lysine is probably caused by hindered substrate diffusion. Spherical particles of 100 nm and larger diameters were observed by transmission electron microscopy and atomic force microscope techniques after modification of GAPDH with different homocysteine thiolactone concentrations. In case of LDH, branched fibril-like aggregates were observed under the same conditions. Interestingly, crosslinked samples of both proteins were found to have reversible thermal denaturation profiles, indicating that modification with homocysteine thiolactone stabilizes the spatial structure of these enzymes.
Total Chemical Synthesis of an Orf Virus Protein, ORFV002, an Inhibitor of the Master Gene Regulator NF-κB.
Son SJ, Harris PW, Squire CJ … +3 more , Baker EN, Kent SB, Brimble MA
Biopolymers · 2014 Mar · PMID 26820014 · Publisher ↗
ORFV002 is a novel orf viral protein (117 Aa) that inhibits nuclear events through the regulation of the transcriptional activity of NF-κB, a master regulator of human gene expression (Diel et al., J Virol 2011, 85, 264-...
ORFV002 is a novel orf viral protein (117 Aa) that inhibits nuclear events through the regulation of the transcriptional activity of NF-κB, a master regulator of human gene expression (Diel et al., J Virol 2011, 85, 264-275). It is identified as the first nuclear inhibitor of NF-κB produced by orf virus (ORFV) and no homologues in other genera of the Chordopoxvirinae subfamily have been reported to date (Diel et al., J Virol 2011, 85, 264-275). Our molecular structure predictions suggest that ORFV002 may mimic part of IκB, an inhibitor and natural human partner of NF-κB. Recent advances in total chemical synthesis of proteins have provided solutions in overcoming challenges of current recombinant methods of protein isolation for structure elucidation. Aided by Boc solid phase peptide synthesis and native chemical ligation, ORFV002 was successfully synthesized in multimilligram amounts in good yield and high purity.
Antimicrobial peptides against Pseudomonas syringae pv. actinidiae and Erwinia amylovora: Chemical synthesis, secondary structure, efficacy, and mechanistic investigations.
Cameron A, De Zoysa GH, Sarojini V
Biopolymers · 2014 Jan · PMID 24122768 · Publisher ↗
We report on structurally modified dodecapeptide amides (KYKLFKKILKFL-NH2) and two analogs of a hexapeptide amide (WRWYCR-NH2) with antibacterial activity against the Gram negative pathogens Pseudomonas syringae pv. acti...
We report on structurally modified dodecapeptide amides (KYKLFKKILKFL-NH2) and two analogs of a hexapeptide amide (WRWYCR-NH2) with antibacterial activity against the Gram negative pathogens Pseudomonas syringae pv. actinidiae (Psa) and Erwinia amylovora (Ea). Dodecapeptide minimal inhibitory concentrations (MICs) ranged from 3.2 to 15.4 µM, with the unmodified peptide being the most potent against both pathogens. The unmodified dodecapeptide also had 32-58% α-helicity in membrane mimetic environments (50% v/v trifluoroethanol and 30 mM SDS micelles). Structural modifications which included branching, acylation, and conjugation with 5-nitro-2-furaldehyde (NFA) proved detrimental to both antimicrobial activity and α-helicity. Scanning electron microscopy (SEM) revealed distinct morphological changes to bacterial cells treated with the different peptides, leading to blistering of the membrane and cell lysis. MICs of the hexapeptide amide were 3.9-7.7 µM against both pathogens. The hexapeptide acid did not show anti-bacterial activity against either pathogen. However, the NFA conjugated hexapeptide acid was more active than the parent peptide or NFA alone with MICs of 1.6-3.2 µM against the pathogens. SEM analysis revealed shriveling and collapse of bacterial cells treated with the hexapeptide, whereas shortening and compactness on exposure to streptomycin. A colorimetric assay demonstrated that the dodecapeptides were likely to act by targeting the bacterial membrane, whereas the hexapeptides, streptomycin, and NFA were not, thereby supporting the morphological changes observed during SEM. To the best of our knowledge, this appears to be the first report of antimicrobial peptide activity against Psa, a pathogen that is currently devastating the kiwifruit industry internationally.
Design and synthesis of α-conotoxin GID analogues as selective α4β2 nicotinic acetylcholine receptor antagonists.
Banerjee J, Yongye AB, Chang YP … +3 more , Gyanda R, Medina-Franco JL, Armishaw CJ
Biopolymers · 2014 Jan · PMID 24122487 · Publisher ↗
The α4β2 nicotinic acetylcholine receptor (nAChR) is an important target for currently approved smoking cessation therapeutics. However, the development of highly selective α4β2 nAChR antagonists remains a significant ch...
The α4β2 nicotinic acetylcholine receptor (nAChR) is an important target for currently approved smoking cessation therapeutics. However, the development of highly selective α4β2 nAChR antagonists remains a significant challenge. α-Conotoxin GID is an antagonist of α4β2 nAChRs, though it is significantly more potent toward the α3β2 and α7 subtypes. With the goal of obtaining further insights into α-conotoxin GID/nAChR interactions that could lead to the design of GID analogues with improved affinity for α4β2 nAChRs, we built a homology model of the GID/α4β2 complex using an X-ray co-crystal structure of an α-conotoxin/acetylcholine binding protein (AChBP) complex. Several additional interactions that could potentially enhance the affinity of GID for α4β2 nAChRs were observed in our model, which led to the design and synthesis of 22 GID analogues. Seven analogues displayed inhibitory activity toward α4β2 nAChRs that was comparable to GID. Significantly, both GID[A10S] and GID[V13I] demonstrated moderately improved selectivity toward α4β2 over α3β2 when compared with GID, while GID[V18N] exhibited no measurable inhibitory activity for the α3β2 subtype, yet retained inhibitory activity for α4β2. In this regard, GID[V18N] is the most α4β2 nAChR selective α-conotoxin analogue identified to date.
A serendipitous survey of prediction algorithms for amyloidogenicity.
Roland BP, Kodali R, Mishra R … +1 more , Wetzel R
Biopolymers · 2013 Nov · PMID 23893755 · Full text
The 17- amino acid N-terminal segment of the Huntingtin protein, htt(NT), grows into stable α-helix rich oligomeric aggregates when incubated under physiological conditions. We examined 15 scrambled sequence versions of...
The 17- amino acid N-terminal segment of the Huntingtin protein, htt(NT), grows into stable α-helix rich oligomeric aggregates when incubated under physiological conditions. We examined 15 scrambled sequence versions of an htt(NT) peptide for their stabilities against aggregation in aqueous solution at low micromolar concentration and physiological conditions. Surprisingly, given their derivation from a sequence that readily assembles into highly stable α-helical aggregates that fail to convert into β-structure, we found that three of these scrambled peptides rapidly grow into amyloid-like fibrils, while two others also develop amyloid somewhat more slowly. The other 10 scrambled peptides do not detectibly form any aggregates after 100 h incubation under these conditions. We then analyzed these sequences using four previously described algorithms for predicting the tendencies of peptides to grow into amyloid or other β-aggregates. We found that these algorithms-Zyggregator, Tango, Waltz, and Zipper-varied greatly in the number of sequences predicted to be amyloidogenic and in their abilities to correctly identify the amyloid forming members of this scrambled peptide collection. The results are discussed in the context of a review of the sequence and structural factors currently thought to be important in determining amyloid formation kinetics and thermodynamics.
NMR-profiles of protein solutions.
Pedrini B, Serrano P, Mohanty B … +2 more , Geralt M, Wüthrich K
Biopolymers · 2013 Nov · PMID 23839514 · Full text
NMR-Profiles are quantitative one-dimensional (1D) presentations of 2D [¹⁵N, ¹H]-correlation spectra used to monitor the quality of protein solutions prior to and during NMR structure determinations and functional studie...
NMR-Profiles are quantitative one-dimensional (1D) presentations of 2D [¹⁵N, ¹H]-correlation spectra used to monitor the quality of protein solutions prior to and during NMR structure determinations and functional studies. In our current use in structural genomics projects, an NMR-Profile is recorded at the outset of a structure determination, using a uniformly ¹⁵N-labeled microscale sample of the protein. We thus assess the extent to which polypeptide backbone resonance assignments can be achieved with given NMR techniques, for example, conventional triple resonance experiments or APSY-NMR. With the availability of sequence-specific polypeptide backbone resonance assignments in the course of the structure determination, an "Assigned NMR-Profile" is generated, which visualizes the variation of the ¹⁵N - ¹H correlation cross peak intensities along the sequence and thus maps the sequence locations of polypeptide segments for which the NMR line shapes are affected by conformational exchange or other processes. The Assigned NMR-Profile provides a guiding reference during later stages of the structure determination, and is of special interest for monitoring the protein during functional studies, where dynamic features may be modulated during physiological processes.
Strategies for profiling native S-nitrosylation.
Majmudar JD, Martin BR
Biopolymers · 2014 Feb · PMID 23828013 · Full text
Cysteine is a uniquely reactive amino acid, capable of undergoing both nucleophlilic and oxidative post-translational modifications. One such oxidation reaction involves the covalent modification of cysteine via the gase...
Cysteine is a uniquely reactive amino acid, capable of undergoing both nucleophlilic and oxidative post-translational modifications. One such oxidation reaction involves the covalent modification of cysteine via the gaseous second messenger nitric oxide (NO), termed S-nitrosylation (SNO). This dynamic post-translational modification is involved in the redox regulation of proteins across all phylogenic kingdoms. In mammals, calcium-dependent activation of NO synthase triggers the local release of NO, which activates nearby guanylyl cyclases and cGMP-dependent pathways. In parallel, diffusible NO can locally modify redox active cellular thiols, functionally modulating many redox sensitive enzymes. Aberrant SNO is implicated in the pathology of many diseases, including neurodegeneration, inflammation, and stroke. In this review, we discuss current methods to label sites of SNO for biochemical analysis. The most popular method involves a series of biochemical steps to mask free thiols followed by selective nitrosothiol reduction and capture. Other emerging methods include mechanism-based phosphine probes and mercury enrichment chemistry. By bridging new enrichment approaches with high-resolution mass spectrometry, large-scale analysis of protein nitrosylation has highlighted new pathways of oxidative regulation.
Rearranging RNA structures at 75°C? Toward the molecular mechanism and physiological function of the Thermus thermophilus DEAD-box helicase Hera.
Klostermeier D
Biopolymers · 2013 Dec · PMID 23765433 · Publisher ↗
DEAD-box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Hera is a DEAD-box helicase from Thermus thermophilus that consists of a helicase core, followed by a C-terminal extension comprising a dimer...
DEAD-box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Hera is a DEAD-box helicase from Thermus thermophilus that consists of a helicase core, followed by a C-terminal extension comprising a dimerization domain and an RNA-binding domain. The combined structural information on individual Hera domains provides a molecular model of the Hera dimer. The modular architecture with flexible connections between individual domains affords different relative orientations of the RBD relative to the Hera helicase core, and of the two helicase cores within the dimer. Presumably, domain movements are intimately linked to RNA binding, to the interplay of the RBD and the helicase core, and to RNA unwinding, and may impact on the functional cooperation of the two helicase cores in RNA unwinding. The in vivo function of Hera is unknown. The Hera RBD recognizes two distinct elements in the RNA substrate, a single-stranded and a structured region. The helicase core then unwinds an adjacent RNA duplex in an ATP-dependent reaction. Overall, this mode of action is reminiscent of DEAD-box proteins that act as general RNA chaperones. This review summarizes the current knowledge on Hera structure and function, and discusses a possible role of Hera in the Thermus thermophilus cold-shock response.
Steric and electronic interactions controlling the cis/trans isomer equilibrium at X-Pro tertiary amide motifs in solution.
Reddy DN, Prabhakaran EN
Biopolymers · 2014 Jan · PMID 23653336 · Publisher ↗
A systematic understanding of the noncovalent interactions that influence the structures of the cis conformers and the equilibrium between the cis and the trans conformers, of the X-Pro tertiary amide motifs, is presente...
A systematic understanding of the noncovalent interactions that influence the structures of the cis conformers and the equilibrium between the cis and the trans conformers, of the X-Pro tertiary amide motifs, is presented based on analyses of (1)H-, (13)C-NMR and FTIR absorption spectra of two sets of homologous peptides, X-Pro-Aib-OMe and X-Pro-NH-Me (where X is acetyl, propionyl, isobutyryl and pivaloyl), in solvents of varying polarities. First, this work shows that the cis conformers of any X-Pro tertiary amide motif, including Piv-Pro, are accessible in the new motifs X-Pro-Aib-OMe, in solution. These conformers are uniquely observable by FTIR spectroscopy at ambient temperatures and by NMR spectroscopy from temperatures as high as 273 K. This is made possible by the persistent presence of n(i-1) →π(i)* interactions at Aib, which also influence the disappearance of steric effects at these cis X-Pro rotamers. Second, contrary to conventional understanding, the energy contribution of steric effects to the cis/trans equilibrium at the X-Pro motifs is found to be nonvariant (0.54 ± 0.02 kcal/mol) with increase in steric bulk on the X group. Third, the current studies provide direct evidence for the weak intramolecular interactions namely the n(i-1) →π(i)*, the N(Pro) •••H(i+1) (C5a), and the C7 hydrogen bond that operate and influence the structures, stabilities, and dynamics between different conformational states of X-Pro tertiary amide motifs. NMR and IR spectral data suggest that the cis conformers of X-Pro motifs are ensembles of short-lived rotamers about the C'(X)-N(Pro) bond.
About
- Frequency
- Sun
- Papers found
- 168
- RSS feed
- Subscribe