Protein alignment using cellulose nanocrystals: practical considerations and range of application

Cellulose nanocrystals (CNCs) form liquid crystals in aqueous solution that confer alignment to macromolecules and permit the measurement of residual dipolar couplings. CNCs possess many attractive features as an alignment medium. They are inexpensive, non-toxic, chemically inert, and robust to denaturants and temperature. Despite these advantages, CNCs are seldom employed as an alignment medium and the range of their applicability has not yet been explored. We have re-examined the use of CNCs in biomolecular NMR by analyzing the effects concentration, ionic strength, and temperature on molecular alignment. Stable alignment was obtained over wide ranges of temperature (10–70°C) and pH (2.5–8.0), which makes CNCs potentially very useful in studies of thermophilic proteins and acid-stabilized molecules. Notably, we find that CNC suspensions are very sensitive to the concentrations of biological buffers, which must be taken into account when they are used in NMR analyses. These results have led us to develop a general procedure for preparing aligned samples with CNCs. Using the SH3 domain from the Fyn tyrosine kinase as a model system, we find that CNCs produce an alignment frame collinear with that of the commonly used Pf1 bacteriophage alignment medium, but of opposite magnitude.     

Pattern recognition methods for prediction of helical region in proteins

An approach is described to the problem of predicting α-helical regions in proteins, based on some concepts of pattern recognition theory. It is shown that for each protein there are functions (signs) of the amino acid sequences of protein segments which permit the identification of α-helical or non-α-helical segments. Rules to recognize useful signs have been formulated.     

A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed

Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3–5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.Proteomics experiments typically involve the analysis of peptide mixtures obtained by the enzymatic digestion of proteomes that can be as complex as complete cell lysates (1, 2). Dynamic range of peptide abundances and the sheer number of peptides encountered in these mixtures require extremely sensitive and fast peptide detection and fragmentation (3). Although a first comprehensively identified and quantified proteome has recently been reported (4), further gains in instrumental performance are clearly needed to reduce overall measurement time, improve sequence coverage of identified proteins, and for the in-depth analysis of mammalian proteomes.Among many different instrumental formats (5), the combination of a linear ion trap (6) with a Fourier transform (FT)1 mass spectrometer has rapidly become a popular technological platform in proteomics because it combines the sensitivity, speed, and robustness of ion traps with the high resolution capabilities of FT instruments. The first implementation of this principle used an ion cyclotron resonance instrument with a 7T magnet as the high resolution device (7). Later, the Orbitrap^TM analyzer developed by Makarov was coupled to the LTQ, combining the linear ion trap with a very small and powerful analyzer (8–11).Here we describe a next generation linear ion trap-Orbitrap instrument with significant improvements in ion source transmission and with a new ion trap configuration. We show that this instrument, termed the LTQ Orbitrap Velos, is capable of much higher scan speeds compared with the current LTQ Orbitrap. Furthermore, we implemented more efficient ion extraction for the higher-energy collisional dissociation (HCD) cell (12). Due to this improvement and the 10-fold higher transmission of ions from atmosphere, high resolution and high mass accuracy MS/MS can now routinely be obtained at very high sensitivity and at scan speeds of up to 5 Hz acquisition rates. A related instrument, the LTQ-Velos, which does not contain the Orbitrap analyzer for high resolution measurements, has been described very recently (13).     

The ferrous-dioxygen intermediate in human cytochrome P450 3A4. Substrate dependence of formation and decay kinetics

The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of approximately 30 ms at 5 degrees C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.     

Mapping of antigenic determinants of hepatitis C virus proteins using phage display

Analysis of the properties for individual hepatitis C virus (HCV) proteins makes it possible to establish their molecular structure and conformation, to localize antigenic and immunogenic determinants, to identify protective epitopes, and to solve applied problems (e.g., design of diagnostic tests, vaccines, and drugs). Linear and conformational epitopes of HCV proteins were localized using the phage display technique, and the peptides exposed on the phages selected with monoclonal antibodies against HCV proteins were tested for immunogenicity. Of the 11 epitopes revealed, three were strongly linear; two depended on the secondary; and one on the tertiary structure of the corresponding protein (conformational epitopes). Amino acid sequences involved in the other epitopes were established. The results can be used to improve the diagnosis of hepatitis C, to study the effect of amino acid substitutions on the antigenic properties of HCV proteins, and to analyze the immune response in patients infected with genotypically different HCV. It was shown with the example of the NS5A epitope that phage particles with epitope-mimicking peptides (mimotopes) induce production of antibodies against the corresponding HCV proteins.     

Study of conformation structure and molecular interactions of rosamycin by infrared spectrum analysis

IR spectra of rosamycin and its solutions in inert (CCl4 and C2Cl4), proton acceptor (tetrahydrofuran, hexametapol and diethylamine) and proton donor (CHCl3 and CH3OD) solvents were studied at various concentrations (0.1 to 0.001 mol/l) and temperatures (20 to 100 degrees C) in the region of the vC = O and vOH absorption bands (1600-1800 and 3200 3650 sm 1). It was found that the absorption bands at 3480 and 3560 sm-1 observed in the spectra of rosamycin diluted solutions in the inert solvents referred to variations of vOH...N of the aminosugar fragment and to vOH...O = C of the ester group of the macrocycle. Bands at 1697 and 1717 sm-1 referred to vC = O of the ketone and aldehyde carbonyl groups and band at 1728 sm-1 referred to vC = O of the ester group whose carbonyl was involved in the C = H...HO intramolecular hydrogen bond. Intensity of vC = O band (1745 sm-1) of the free ester group was nought. However, it increased with using the proton acceptor solvents. OH...N and OH...O = C intramolecular hydrogen bonds stabilized rosamycin molecule conformation. Mechanism of rosamycin interaction with the proton donor and acceptor molecules was elucidated. It was shown that tertiary nitrogen was the center of rosamycin molecule protonation.     

Consensus generation and variant detection by Celera Assembler

Motivation: We present an algorithm to identify allelic variationgiven a Whole Genome Shotgun (WGS) assembly of haploid sequences,and to produce a set of haploid consensus sequences rather thana single consensus sequence. Existing WGS assemblers take acolumn-by-column approach to consensus generation, and producea single consensus sequence which can be inconsistent with theunderlying haploid alleles, and inconsistent with any of thealigned sequence reads. Our new algorithm uses a dynamic windowingapproach. It detects alleles by simultaneously processing theportions of aligned reads spanning a region of sequence variation,assigns reads to their respective alleles, phases adjacent variantalleles and generates a consensus sequence corresponding toeach confirmed allele. This algorithm was used to produce thefirst diploid genome sequence of an individual human. It canalso be applied to assemblies of multiple diploid individualsand hybrid assemblies of multiple haploid organisms. Results: Being applied to the individual human genome assembly,the new algorithm detects exactly two confirmed alleles andreports two consensus sequences in 98.98% of the total number2 033 311 detected regions of sequence variation. In 33 269out of 460 373 detected regions of size >1 bp, it fixes theconstructed errors of a mosaic haploid representation of a diploidlocus as produced by the original Celera Assembler consensusalgorithm. Using an optimized procedure calibrated against 1506 344 known SNPs, it detects 438 814 new heterozygous SNPswith false positive rate 12%. Availability: The open source code is available at: http://wgs-assembler.cvs.sourceforge.net/wgs-assembler/ Contact: gdenisov{at}jcvi.org Associate Editor: John Quackenbush