Ion‐specific modulation of protein interactions: Anion‐induced,reversible oligomerization of a fusion protein

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s_w) and hydrodynamic diameter (D_H) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s_w and D_H, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D_H and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M_z) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I⁻ > Br⁻ > Cl⁻ > F⁻) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.     

Self-association and solubility of peptides: solvent-titration study of N alpha-protected C-terminal sequences of substance P

Self-association of N^α-protected peptides related to C-terminal sequences of substance P in methylene chloride was disrupted by adding increasing amounts of various polar organic solvents. This process was monitored by the disappearance of the amide I C?O stretching band (1630 cm^?1) of strongly intermolecularly H-bonded molecules in the irabsorption spectra. The effects induced by main-chain length, incorporation at position 9 of a residue promoting folding (α-aminoisobutyric acid), the nature of solvent, and peptide concentration were established. A corollary ¹H-nmr investigation provided detailed information on the NH protons involved in the self-association process as H-bonding donors. The increasing propensity to aggregate exhibited by these peptides is paralleled by a decrease in their solubility. The impact of these results on the synthesis of substance P short sequences is briefly outlined.     

Exclusion chromatography of concentrated hemoglobin solutions: Comparison of the self-association behavior of the oxy and deoxy forms of the α2β2 species

Theory pertaining to the interpretation of partition chromatography results obtained with self-associating protein systems studied at high total concentrations is extended to permit consideration of situations in which both monomeric and dimeric states partition. This development, which includes considerations of thermodynamic nonideality effects, permits a quantitative correlation of human oxyhemoglobin results reported previously and obtained in this work employing a different stationary matrix of controlled-pore glass beads. The two sets of results, obtained at pH 7.3 and 20°C- indicate that the α₂β₂ species of oxyhemoglobin self-associates. Two types of association pattern, discrete dimerization and an indefinite self-association, are examined. This is done for a realistic range of values for the radius, r, of the effective hard sphere appropriate to the calculation of the covolume of the α₂β₂ species in the assessment of the thermodynamic nonideality contribution. Assessed values of the isodesmic association constant range from 66 = 23 M ^?1(r = 2.84 nm) to 154 = 26 M^?1' (r = 3.13 nm). This mode of indefinite association is marginally favored over a dimerization when the larger value of r is considered, the two patterns becoming virtually indistinguishable for the lower value of r. Partition chromatography results are also presented for human deoxyhemoglobin up to a total concentration of 225 $\text{g}\text{I}$, and are analyzed in a similar fashion to show that the indefinite self-association pattern is favored, governed by an isodesmic constant in the range 91 = 9 M^?1(r = 2.84 nm) to 223 = 84 M^?1 (r = 3.13 nm). Comparison of the constants assessed for the oxy and deoxy systems permits discussion of the concept that oxygen binds preferentially to the α₂β₂ species of deoxyhemoglobin in comparison with its polymers.     

De novo design of transmembrane helix–helix interactions and measurement of stability in a biological membrane

Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu “scaffold” was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix–helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔG^app) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (< 1.4 kcal mol^− 1) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes).     

Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association

Calmodulin (CaM)-kinase II holoenzymes composed of either alpha or beta subunits were analyzed using light scattering to determine a mechanism for self-association. Under identical reaction conditions, only alphaCaM-kinase II holoenzymes self-associated. Self-association was detected at a remarkably low enzyme concentration (0.14 microM or 7 microg/mL). Light scattering revealed two phases of self-association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2-3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self-association required activation by Ca(2+)/CaM and was strongly dependent on pH. Self-association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide-2) and inhibitor (AIP) designed from the autoregulatory domain of CaM-kinase II potently prevented self-association, whereas the peptide substrate syntide-2 did not. Thus, CaM-kinase II self-association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory-like sequence. A model for CaM-kinase II self-association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit-interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM-kinase II previously reported in models of ischemia.     

Screening of a Small Set of Random Peptides: A New Strategy to Identify Synthetic Peptides that Mimic Epitopes

Small diversity libraries, composed of 4550 synthetic dodecapeptides and 8000 synthetic tripeptides, have been used to identify sequences homologous to small linear and non-linear parts of epitopes. Here we report that synthetic peptides identified through alignment of dodecapeptides and tripeptides derived from these small libraries have, in direct ELISA and/or competitive ELISA, activities similar to that of peptides covering the native epitope and similar to that of peptides derived from large expression libraries composed of 10⁶–10⁷ random peptides. This result was obtained with the monoclonal antibodies 6A.A6 and M2. Mab 6A.A6 binds the transmissible gastroenteritis virus (TGEV) and mAb M2 binds the FLAG^®-peptide, an affinity tag. It was also found that the antibody binding activity of peptides, derived from small or large libraries, can strongly depend on the way in which the peptide is presented to the antibody, i.e. high antibody titers were obtained when these peptides were synthesized on pins or coated onto microtiter plates, whereas low IC₅₀s were obtained with these peptides in solution. We postulate that small peptide libraries may be a powerful tool to quickly identify new peptides that can be used as sensitive markers for mAbs of interest. © 1997 John Wiley & Sons, Ltd.     

Shotgun Protein Sequencing with Meta-contig Assembly

Full-length de novo sequencing from tandem mass (MS/MS) spectra of unknown proteins such as antibodies or proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low sequencing accuracy. Our shotgun protein sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous proteins to group smaller contig sequences into full-length protein sequences, thus limiting its effectiveness on sequences from poorly annotated proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo sequencing limitations relate to MS/MS acquisition settings.Database search tools, such as Sequest (3), Mascot (4), and InsPecT (5), are the most frequently used methods for reliable protein identification in tandem mass (MS/MS) spectrometry based proteomics. These operate by separately matching each MS/MS spectrum to peptide sequences from reference protein databases where all proteins of interest are presumably contained. But this assumption often does not hold true as many important proteins, such as monoclonal antibodies, are not contained in any database because mechanisms of antibody variation (including genetic recombination and somatic hyper-mutation (6)) constantly create new proteins with novel unique sequences. These mechanisms of variation are the foundation of adaptive immune systems and have enabled highly successful antibody-based therapeutic strategies (7, 8). Nevertheless, such variation also means that antibody MS/MS spectra are typically impossible to identify via standard database search techniques whenever the corresponding sequences are not known in advance. An inherent drawback of database search strategies is that they are only as good as the database(s) being searched and incomplete databases often result in proteins being misidentified or left unidentified (9).Despite the importance of novel protein identification, few high-throughput methods have been developed for de novo sequencing of unknown proteins. Low-throughput Edman degradation is a well-known de novo sequencing approach that can accurately call amino acid sequences in N/C-terminal regions of unknown proteins but has drawbacks that make it unsuitable for sequencing proteins longer than 50 amino acids or proteins with post-translational modifications (10, 11). Many have recognized the potential of tandem mass spectrometry for protein sequencing. For example, in 1987 Johnson and Biemann (12) manually sequenced a complete protein from rabbit bone marrow. Meanwhile, automated de novo sequencing methods that rely on interpretations of individual MS/MS spectra are limited in that they typically cannot reconstruct long (8+ AA) sequences without mis-predicting 1 in 5 AA on average for low accuracy collision-induced dissociation (CID) spectra (13, 14). Recent advances in de novo peptide sequencing have improved sequencing accuracy to over 95% for high resolution higher energy collisional dissociation (HCD)¹ spectra (15), but at limited sequence coverage (Chi H et al. report only 55% sequence coverage of peptides identified by database search). In fact, all current per-spectrum de novo sequencing strategies face a significant tradeoff between sequencing accuracy and coverage as spectra exhibiting complete peptide fragmentation rarely cover entire target proteins, yet are required to accurately reconstruct full-length peptide sequences. An alternative approach to separately sequencing individual spectra is to simultaneously interpret multiple MS/MS spectra from overlapping peptides. This Shotgun Protein Sequencing (SPS) paradigm differs from traditional algorithms by deriving consensus sequences from contigs - sets of multiple MS/MS spectra from distinct peptides with overlapping sequences (1, 16). Because SPS aggregates multiple spectra from overlapping peptides, protein sequences extending beyond the length of enzymatically digested peptides can be extracted from spectra with incomplete peptide fragmentation. Furthermore, SPS has been found to generate sequences that frequently cover 90–95+% of the target protein sequence(s) whereas mis-predicting only 1 out of every 20 amino acids on high resolution MS/MS spectra (2). But a remaining limitation of SPS is that it still generates fragmented sequences that do not singularly cover large regions of the target protein sequences, much less complete proteins: SPS sequences have an average length of 10–15 amino acids (depending on input data) and the longest recovered SPS de novo sequence is less than 45 amino acids long (1).The considerable limitations of de novo sequencing strategies have typically been addressed by attempting to circumvent them using error-tolerant matching to known protein sequences. One such strategy (17) is to generate short de novo sequence tags and then match them exactly to protein databases without requiring matching the N/C-term flanking masses (to allow for unexpected polymorphisms or post-translational modifications). Short sequence tags are usually derived from parts of the spectrum with high signal-to-noise ratios and typically have higher sequencing accuracy than full-length de novo sequences (18). This approach was later extended in MS-Shotgun (19) and continues to be a popular technique for speeding up database search tools (5, 20–22). Homology matching of full length de novo sequences was first explored in CIDentify (23) and later in MS-BLAST (24) by searching de novo sequences using FASTA and WU-BLAST2 (respectively) to find homologous matches to sequences of related proteins; FASTS (25) also approached the problem using a modified version of FASTA. However, common de novo sequencing errors tend to produce sequences that are heavily penalized in pure sequence homology searches. For example, missing peaks in MS/MS spectra may easily cause GA subsequences to be reconstructed as Q or AG (same-mass sequences), thus making subsequent BLAST searches unlikely to succeed. This issue was partially considered in CIDentify and more thoroughly addressed in SPIDER (26) by explicitly modeling de novo sequencing errors together with BLOSUM scores in MS/MS-based sequence homology searches. In addition, OpenSea (27) further explored database matching of de novo sequences for analysis of unexpected post-translational modifications (PTMs). Finally, Shen et al. (28) used short unique de novo sequence tags, called UStags, to discover protein-localized PTMs.Recent approaches to homology matching of de novo sequences have built on genome assembly and sequencing techniques to achieve database-assisted full-length sequencing of unknown proteins. Comparative Shotgun Protein Sequencing (cSPS) complemented SPS assembly techniques with usage of error tolerant matching of de novo sequences to find overlapping SPS de novo sequences that are then further assembled into full-length protein sequences (2). cSPS was designed to support the sequencing of highly divergent proteins that have regions close enough in homology to transfer matches from a reference. cSPS was shown to enable de novo sequencing of monoclonal antibodies at 95+% sequencing accuracy, while simultaneously tolerating and identifying unexpected PTMs (29). In difference from cSPS, Champs (30) de novo sequences individual spectra to obtain putative peptide sequences, which are then mapped to homologous proteins to correct sequencing errors and reconstruct protein sequences with 100% accuracy and 99% coverage. However, Champs is designed to only map peptides that differ from the reference sequence by one or two amino acids and does not handle PTMs. As such, its sequencing accuracy is not directly comparable to that of cSPS as Champs was not designed to sequence highly divergent proteins (such as monoclonal antibodies) with multiple PTMs, insertions, deletions, and/or recombinations. GenoMS (31) extended the approaches in cSPS/Champs by explicitly modeling protein splice variants as paths in splice graphs where nodes represent translated exon regions (32). MS/MS spectra are first searched for exact sequence matches against all possible protein isoforms. The remaining unidentified MS/MS spectra are then aligned to the matched peptides and de novo sequenced to extend the matched sequences into novel regions. Reported sequences are 97–99% accurate and cover 96–99% of target proteins depending on sequence similarity between the novel and reference sequences (31). However, GenoMS de novo sequences are usually extended less than 3 amino acids beyond matched peptides because sequencing accuracy degrades as sequences are extended, thus preventing the consistent extension of long (10+ AA) sequences. Altogether, the use of homology matching approaches for full-length de novo protein sequencing continues to be limited by 1) requiring the previous knowledge of closely related protein sequences and 2) the inherent difficulties in statistically significant homology-tolerant matching of error-prone short de novo sequences.The Meta-SPS approach proposed here seeks to de novo sequence complete proteins, or long protein regions, without any use of a database. Meta-SPS builds upon SPS by treating SPS de novo sequences (contig sequences) as input spectra and further assembling them into longer de novo sequences (meta-contig sequences). We show that Meta-SPS extends de novo sequences to lengths over 100 AA while boosting sequencing accuracy to only 1 mistake per 40 amino acid predictions, thus enabling database-free de novo sequencing of completely novel proteins while also allowing error-tolerant matching approaches to support higher-divergence homologies (by searching longer, more accurate de novo sequences). Meta-SPS algorithms are demonstrated on CID and HCD MS/MS spectra and its limitations are discussed in relation to the underlying limitations of bottom-up tandem mass spectrometry.     

Separation of D-amino acid-containing peptide phenylseptin using 3,3′-phenyl-1,1′-binaphthyl-18-crown-6-ether columns

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3′-phenyl-1,1′-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (−), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (−) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH₃⁺ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.     

Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (k_on, association) and reverse (k_off, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′_on = 2.1(1) × 10⁶ M⁻¹ s⁻¹) and 6 bp (k′_on = 5.0(1) × 10⁶ M⁻¹ s⁻¹) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (k_off = 0.024 s⁻¹) to 6 bp (k_off = 14 s⁻¹) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of k_on and k_off is measured. Interestingly, k_onincreases by >40-fold (k_on = 0.10(1) s⁻¹ to 4.0(4) s⁻¹ between [NaCl] = 25 mM and 1 M), whereas in contrast, k_offdecreases by fourfold (0.72(3) s⁻¹ to 0.17(7) s⁻¹) over the same range of conditions. Thus, the equilibrium constant (K_eq) increases by ≈160, largely due to changes in the association rate, k_on. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.     

Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B

The bacterial serine protease, SGPB, was inhibited by two specific tripeptide chloromethyl ketones, N-t-butyloxycarbonyl-l-alanylglycyl-l-phenylalanine chloromethyl ketone (BocAGFCK) and N-t-butyloxycarbonyl-glycyl-l-leucyl-l-phenylalanine chloromethyl ketone (BocGLFCK). Crystals of the inhibited complexes were grown and examined by X-ray crystallographic methods. The peptide backbone of each inhibitor is bound by three hydrogen bonds to the main chain of residues Ser214 to Gly216. There are two well-characterized hydrophobic pockets, S₁ and S₂, on the surface of SGPB which accommodate the P₁ and P₂ side-chains of the BocGLFCK inhibitor. A conformational change of Tyr171 is induced by the binding of this inhibitor. Both inhibitors make two covalent bonds to the SGPB enzyme. The imidazole ring of His57 is alkylated at the N^?2 atom and O^γ of Ser195 forms a hemiketal bond with the carbonyl-carbon atom of the inhibitor. Comparison of the binding modes of the two tripeptides in conjunction with the differences in their inhibition constants (K_I) allows one to estimate the binding energy of the leucyl side-chain as ?2.6 kcal mol^?1. The importance of an electrophilic component in the serine protease mechanism, which involves the polarization of the susceptible carbonyl bond of a substrate or inhibitor by the peptide NH groups of Gly193 and Ser195 is discussed.     

De Novo Purine Biosynthesis in Intact Cells of Cucurbita pepo

The capacity of intact cells of roots excised from summer squash plants (Cucurbita pepo L. cv Early Prolific Straightneck) to synthesize purine nucleotides de novo was investigated. Evidence that purine nucleotides are synthesized de novo included: (a) demonstration of the incorporation of [1-¹⁴C]glycine, [2-¹⁴C]glycine, NaH¹⁴CO₃, and H¹⁴COONa into total adenine nucleotides; (b) observation that the addition of azaserine or aminopterin, known inhibitors of de novo purine synthesis in other organisms, blocked the incorporation of these precursors into adenine nucleotides; and (c) demonstration that the purine ring synthesized from these precursors was labeled in a manner consistent with the pathway for de novo purine biosynthesis found in microorganisms and animal tissues. Under optimal conditions, the activity of this pathway in roots excised from 2-day-old squash plants was 244 ± 13 nanomoles (mean ± standard error, n = 17) NaH¹⁴CO₃ incorporated into ∑Ade (the sum of the adenine nucleotides, nucleoside and free base) per gram tissue during the 3-hour incubation period.

The possible occurrence of alternative enzymic reactions for the first steps of de novo purine biosynthesis was also investigated. No conclusive evidence was obtained to support the operation of alternative enzymic reactions in the intact cell of C. pepo.

     

Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)₂(SPA1)₂] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440–669?kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1^L105A (which forms homodimers) and COP1^L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1^L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440–669?kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.     

Enantiomer-selective solvolysis catalysed by a histidine-containing cyclic dipeptide carrying chiral side chain

Tripeptides with cyclic dipeptide backbones, cyclo[l-Glu(l-Leu-O Bzl)-t-His] and cyclo[l-Glu(l-Leu-OH)-Ir His, and the corresponding tripeptides with linear backbones, Me₃COCO-l-Glu(l-Leu-OBzl)-l-His-OMe and Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, were synthesized and used as catalysts for the hydrolysis of carboxylic acid active esters of various types. The experimental results are summarized as follows. (I) In the hydrolysis of a neutral and hydrophobic substate, p-nitrophenyl laurate, in 20% dioxane/H₂O mixture of pH 7.8, a hydrophobic and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. On the other hand, cyclo[l-Glu(l-Leu-OBzl)-l-His] and cyclo[l-Leu-OH)-l-His], which have rigid backbone chain and fixed sidechain conformation, were not particularly reactive. (2) in the solcolysis of a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a negatively charged and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. However, cyclo [l-Glu(l-Leu-OH)-l-His] was not particularly reactive in the same reaction. In the hydrolysis of p-nitrophenyl glycinate hydrochloride in aqueous solution at pH 7.8 a hydrophobic and rigid peptide, cyclo[(l-Glu(l-Leu-OBzl)-l-His], was more reactive than imidazole. However, in the hydrolysis of p-nitrophenyl CO-AMINODODECANOATE hydrochloride, which has a positive charge and a rective site separated by a long hydrophobic chain, peptide catalysts did not show efficient catalysis. (3) In the hydrolysis of a positively charged, hydrophobic and chiral substrate, p-nitrophenyl leucinate hydrochloride, in aqueous solution at pH 6.95, the d-enantiomer was hydrolysed more quickly that the t-enantiomer with cyclo[l-Glu(l-Leu-OBzl)l-His] or cyclo[t-Glu(l-Leu-OH)-l-His] as catalyst. On the other hand, the tripeptides with linear backbone did not effect an enantiomer-selective catalysis. The solvolytic reaction catalysed by the tripeptides with cyclic dipeptide backbone in 42% i-PrOH/water mixture was also enantiomer-selective.     

Characterization of subunit structural alterations which occur during purification of nitrate reductase from Escherichia coli

Nitrate reductase from Escherichia coli, purified to homogeneity after release from membranes by deoxycholate treatment, was composed of two subunits of 155,000 (α) and 58,000 (β) daltons and contained no cytochrome b₁. Analysis of fractions at different stages of purification by gel electrophoresis and immunoprecipitation revealed that during the early steps of the purification cytochrome b₁ dissociated from the enzyme and the β subunit was altered in size as determined by sodium dodecyl sulfate-gel electrophoresis. Analysis of the peptide patterns obtained by partial proteolysis of isolated α and β subunits established that these subunits are composed of distinct sequences and ruled out a precursor-product relationship between the two subunits. The β subunit was altered during the purification by loss of a 2000-dalton fragment, apparently from its carboxyl terminus. The protease inhibitor tosyllysine chloromethylketone protected nitrate reductase from more extensive degradation by endogenous proteases during the purification but did not prevent the removal of the 2000-dalton fragment. This carboxyl terminal fragment was part of a 15,000-dalton sequence which was removed by trypsin and which was required for the self-associating character of the unmodified enzyme monomers. From the structural changes which occurred during the purification procedure, it is proposed that the carboxyl terminal segment of the β subunit is involved in the binding of nitrate reductase to cytochrome b₁ and its association with the membrane.     

On the design and efficacy assessment of self‐assembling peptide‐based hydrogel‐glycosaminoglycan mixtures for potential repair of early stage cartilage degeneration

Peptide‐based hydrogels are of interest for their potential use in regenerative medicine. Combining these hydrogels with materials that may enhance their physical and biological properties, such as glycosaminoglycans, has the potential to extend their range of biomedical applications, for example in the repair of early cartilage degeneration. The aim of this study was to combine three self‐assembling peptides (P₁₁‐4, P₁₁‐8, and P₁₁‐12) with chondroitin sulphate at two molar ratios of 1:16 and 1:64 in 130 and 230 mM Na⁺ salt concentrations. The study investigates the effects of mixing self‐assembling peptide and glycosaminoglycan on the physical and mechanical properties at 37°C. Peptide alone, chondroitin sulphate alone, and peptide in combination with chondroitin sulphate were analysed using Fourier transform infrared spectroscopy to determine the β‐sheet percentage, transmission electron microscopy to determine the fibril morphology, and rheology to determine the elastic and viscous modulus of the materials. All of the variables (peptide, salt concentration, and chondroitin sulphate molar ratio) had an effect on the mechanical properties, β‐sheet formation, and fibril morphology of the hydrogels. P₁₁‐4 and P₁₁‐8‐chondroitin sulphate mixtures, at both molar ratios, were shown to have a high β‐sheet percentage, dense entangled fibrillar networks, as well as high mechanical stiffness in both (130 and 230 mM) Na⁺ salt solutions when compared with the P₁₁‐12/chondroitin sulphate mixtures. These peptide/chondroitin sulphate hydrogels show promise for biomedical applications in glycosaminoglycan depleted tissues.     

Enhanced De Novo Assembly of High Throughput Pyrosequencing Data Using Whole Genome Mapping

Despite major advances in next-generation sequencing, assembly of sequencing data, especially data from novel microorganisms or re-emerging pathogens, remains constrained by the lack of suitable reference sequences. De novo assembly is the best approach to achieve an accurate finished sequence, but multiple sequencing platforms or paired-end libraries are often required to achieve full genome coverage. In this study, we demonstrated a method to assemble complete bacterial genome sequences by integrating shotgun Roche 454 pyrosequencing with optical whole genome mapping (WGM). The whole genome restriction map (WGRM) was used as the reference to scaffold de novo assembled sequence contigs through a stepwise process. Large de novo contigs were placed in the correct order and orientation through alignment to the WGRM. De novo contigs that were not aligned to WGRM were merged into scaffolds using contig branching structure information. These extended scaffolds were then aligned to the WGRM to identify the overlaps to be eliminated and the gaps and mismatches to be resolved with unused contigs. The process was repeated until a sequence with full coverage and alignment with the whole genome map was achieved. Using this method we were able to achieved 100% WGRM coverage without a paired-end library. We assembled complete sequences for three distinct genetic components of a clinical isolate of Providencia stuartii: a bacterial chromosome, a novel bla _NDM-1 plasmid, and a novel bacteriophage, without separately purifying them to homogeneity.     

Sedimentation coefficients of self-associating species: I. Basic theory

Under the same solution conditions, the apparent weight average sedimentation coefficient, s_wa, and some quantities obtained from it can be combined with the equilibrium constant or constants, K_i, and the monomer concentration, c_I, obtained from sedimentation equilibrium, light scattering or osmotic pressure experiments on the same self-associating solute, so that the individual sedimentation coefficients, s_i, of the self-associating species, and also the hydrodynamic concentration dependence parameter,g or $\text{g}$, can be evaluated. Using two different models for the hydrodynamic concentration parameter, four different methods are presented for the evaluation of the s_i's. Methods for evaluating g or $\text{g}$, once the s_i's are known, are presented. A method for obtaining the number average sedimentation coefficient, s_N, and its application to self-associations is presented. Methods are shown for the evaluation of Z average properties, x_zc, as well as number average properties,x_Nc, of a self-associating solute from its weight average properties, x_wc.     

Linear oligopeptides: peptaibol antibiotics — preferred conformation of the 2–9 segment of emerimicins III and IV and all related short sequences

With the aim of obtaining information on the effect induced by main-chain length and amino acid sequence on the type of helical structure adopted by naturally-occurring peptides rich in C^α,α-dialkylated residues, an infrared absorption and ¹H nuclear magnetic resonance analysis of chloroform solutions of the protected 2–9 segment of the peptaibol antibiotics emerimicins III and IV,-(Aib)₃-l-Val-Gly-l-Leu-(Aib)₂-, and all related short sequences starting from both the N- and C-termini was performed. The results are consistent with the presence of folded structures of the β-bend type (in the shorter peptides) or 3₁₀-helices (in the longer peptides). Extent of formation and stability of the inter- and intramolecular H-bonds have been assessed as a function of concentration, temperature, addition of dimethylsulphoxide (DMSO) and the free radical 2,2,6,6-tetramethy-1-piperidinyloxy (TEMPO). At high peptide concentration both folded and helical structures tend to self-associate extensively. In the self-association process the N(1)H and N(2)H groups are those acting as H-bonding donors. These results agree well with those obtained in the solid state by X-ray diffraction on the octapeptide itself and selected short sequences.