A revision of chiggers of the <Emphasis Type="Italic">minuta</Emphasis> species-group (Acari: Trombiculidae: <Emphasis Type="Italic">Neotrombicula</Emphasis> Hirst, 1925) using multivariate morphometrics

We revise chiggers belonging to the minuta-species group (genus Neotrombicula Hirst, 1925) from the Palaearctic using size-free multivariate morphometrics. This approach allowed us to resolve several diagnostic problems. We show that the widely distributed Neotrombicula scrupulosa Kudryashova, 1993 forms three spatially and ecologically isolated groups different from each other in size or shape (morphometric property) only: specimens from the Caucasus are distinct from those from Asia in shape, whereas the Asian specimens from plains and mountains are different from each other in size. We developed a multivariate classification model to separate three closely related species: N. scrupulosa, N. lubrica Kudryashova, 1993 and N. minuta Schluger, 1966. This model is based on five shape variables selected from an initial 17 variables by a best subset analysis using a custom size-correction subroutine. The variable selection procedure slightly improved the predictive power of the model, suggesting that it not only removed redundancy but also reduced ‘noise’ in the dataset. The overall classification accuracy of this model is 96.2, 96.2 and 95.5%, as estimated by internal validation, external validation and jackknife statistics, respectively. Our analyses resulted in one new synonymy: N. dimidiata Stekolnikov, 1995 is considered to be a synonym of N. lubrica. Both N. scrupulosa and N. lubrica are recorded from new localities. A key to species of the minuta-group incorporating results from our multivariate analyses is presented.     

Recovery of tubulin functions after freeze-drying in the presence of trehalose

Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at −80 °C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assembly-competent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.     

Stretched DNA Investigated Using Molecular-Dynamics and Quantum-Mechanical Calculations

We combined atomistic molecular-dynamics simulations with quantum-mechanical calculations to investigate the sequence dependence of the stretching behavior of duplex DNA. Our combined quantum-mechanical/molecular-mechanical approach demonstrates that molecular-mechanical force fields are able to describe both the backbone and base-base interactions within the highly distorted nucleic acid structures produced by stretching the DNA from the 5′ ends, which include conformations containing disassociated basepairs, just as well as these force fields describe relaxed DNA conformations. The molecular-dynamics simulations indicate that the force-induced melting pathway is sequence-dependent and is influenced by the availability of noncanonical hydrogen-bond interactions that can assist the disassociation of the DNA basepairs. The biological implications of these results are discussed.     

Design of Active Transport Must Be Highly Intricate: A Possible Role of Myosin and Ena/VASP for G-Actin Transport in Filopodia

Recent modeling of filopodia—the actin-based cell organelles employed for sensing and motility—reveals that one of the key limiting factors of filopodial length is diffusional transport of G-actin monomers to the polymerizing barbed ends. We have explored the possibility of active transport of G-actin by myosin motors, which would be an expected biological response to overcome the limitation of a diffusion-based process. We found that in a straightforward implementation of active transport the increase in length was unimpressive, ≤30%, due to sequestering of G-actin by freely diffusing motors. However, artificially removing motor sequestration reactions led to approximately threefold increases in filopodial length, with the transport being mainly limited by the motors failing to detach from the filaments near the tip, clogging the cooperative conveyer belt dynamics. Making motors sterically transparent led to a qualitative change of the dynamics to a different regime of steady growth without a stationary length. Having identified sequestration and clogging as ubiquitous constraints to motor-driven transport, we devised and tested a speculative means to sidestep these limitations in filopodia by employing cross-linking and putative scaffolding roles of Ena/VASP proteins. We conclude that a naïve design of molecular-motor-based active transport would almost always be inefficient—an intricately organized kinetic scheme, with finely tuned rate constants, is required to achieve high-flux transport.     

Heterologous expression of leader-less <Emphasis Type="Italic">pga</Emphasis> gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis>: intracellular production of prokaryotic enzyme

Background  

Penicillin G acylase of Escherichia coli (PGA_Ec) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGA_Ec in this host and studied yeast production capacity and enzyme authenticity.     

The Generating Function of CID,ETD, and CID/ETD Pairs of Tandem Mass Spectra: Applications to Database Search

Recent emergence of new mass spectrometry techniques (e.g. electron transfer dissociation, ETD) and improved availability of additional proteases (e.g. Lys-N) for protein digestion in high-throughput experiments raised the challenge of designing new algorithms for interpreting the resulting new types of tandem mass (MS/MS) spectra. Traditional MS/MS database search algorithms such as SEQUEST and Mascot were originally designed for collision induced dissociation (CID) of tryptic peptides and are largely based on expert knowledge about fragmentation of tryptic peptides (rather than machine learning techniques) to design CID-specific scoring functions. As a result, the performance of these algorithms is suboptimal for new mass spectrometry technologies or nontryptic peptides. We recently proposed the generating function approach (MS-GF) for CID spectra of tryptic peptides. In this study, we extend MS-GF to automatically derive scoring parameters from a set of annotated MS/MS spectra of any type (e.g. CID, ETD, etc.), and present a new database search tool MS-GFDB based on MS-GF. We show that MS-GFDB outperforms Mascot for ETD spectra or peptides digested with Lys-N. For example, in the case of ETD spectra, the number of tryptic and Lys-N peptides identified by MS-GFDB increased by a factor of 2.7 and 2.6 as compared with Mascot. Moreover, even following a decade of Mascot developments for analyzing CID spectra of tryptic peptides, MS-GFDB (that is not particularly tailored for CID spectra or tryptic peptides) resulted in 28% increase over Mascot in the number of peptide identifications. Finally, we propose a statistical framework for analyzing multiple spectra from the same precursor (e.g. CID/ETD spectral pairs) and assigning p values to peptide-spectrum-spectrum matches.Since the introduction of electron capture dissociation (ECD)1 in 1998 (1), electron-based peptide dissociation technologies have played an important role in analyzing intact proteins and post-translational modifications (2). However, until recently, this research-grade technology was available only to a small number of laboratories because it was commercially unavailable, required experience for operation, and could be implemented only with expensive FT-ICR instruments. The discovery of electron-transfer dissociation (ETD) (3) enabled an ECD-like technology to be implemented in (relatively cheap) ion-trap instruments. Nowadays, many researchers are employing the ETD technology for tandem mass spectra generation (4–9).Although the hardware technologies to generate ETD spectra are maturing rapidly, software technologies to analyze ETD spectra are still in infancy. There are two major approaches to analyzing tandem mass spectra: de novo sequencing and database search. Both approaches find the best-scoring peptide either among all possible peptides (de novo sequencing) or among all peptides in a protein database (database search). Although de novo sequencing is emerging as an alternative to database search, database search remains a more accurate (and thus preferred) method of spectral interpretation, so here we focus on the database search approach.Numerous database search engines are currently available, including SEQUEST (10), Mascot (11), OMSSA (12), X!Tandem (13), and InsPecT (14). However, most of them are inadequate for the analysis of ETD spectra because they are optimized for collision induced dissociation (CID) spectra that show different fragmentation propensities than those of ETD spectra. Additionally, the existing tandem mass spectrometry (MS/MS) tools are biased toward the analysis of tryptic peptides because trypsin is usually used for CID, and thus not suitable for the analysis of nontryptic peptides that are common for ETD. Therefore, even though some database search engines support the analysis of ETD spectra (e.g. SEQUEST, Mascot, and OMSSA), their performance remains suboptimal when it comes to analyzing ETD spectra. Recently, an ETD-specific database search tool (Z-Core) was developed; however it does not significantly improve over OMSSA (15).We present a new database search tool (MS-GFDB) that significantly outperforms existing database search engines in the analysis of ETD spectra, and performs equally well on nontryptic peptides. MS-GFDB employs the generating function approach (MS-GF) that computes rigorous p values of peptide-spectrum matches (PSMs) based on the spectrum-specific score histogram of all peptides (16).² MS-GF p values are dependent only on the PSM (and not on the database), thus can be used as an alternative scoring function for the database search.Computing p values requires a scoring model evaluating qualities of PSMs. MS-GF adopts a probabilistic scoring model (MS-Dictionary scoring model) described in Kim et al., 2009 (17), considering multiple features including product ion types, peak intensities and mass errors. To define the parameters of this scoring model, MS-GF only needs a set of training PSMs.³ This set of PSMs can be obtained in a variety of ways: for example, one can generate CID/ETD pairs and use peptides identified by CID to form PSMs for ETD. Alternatively, one can generate spectra from a purified protein (when PSMs can be inferred from the accurate parent mass alone) or use a previously developed (not necessary optimal) tool to generate training PSMs. From these training PSMs, MS-GF automatically derives scoring parameters without assuming any prior knowledge about the specifics of a particular peptide fragmentation method (e.g. ETD, CID, etc.) and/or proteolytic origin of the peptides. MS-GF was originally designed for the analysis of CID spectra, but now it has been extended to other types of spectra generated by various fragmentation techniques and/or various enzymes. We show that MS-GF can be successfully applied to novel types of spectra (e.g. ETD of Lys-N peptides (18, 19)) by simply retraining scoring parameters without any modification. Note that although the same scoring model is used for different types of spectra, the parameters derived to score different types of spectra are dissimilar.We compared the performance of MS-GFDB with Mascot on a large ETD data set and found that it generated many more peptide identifications for the same false discovery rates (FDR). For example, at 1% peptide level FDR, MS-GFDB identified 9450 unique peptides from 81,864 ETD spectra of Lys-N peptides whereas Mascot only identified 3672 unique peptides, ≈160% increase in the number of peptide identifications (a similar improvement is observed for ETD spectra of tryptic peptides).⁴ MS-GFDB also showed a significant 28% improvement in the number of identified peptides from CID spectra of tryptic peptides (16,203 peptides as compared with 12,658 peptides identified by Mascot).The ETD technology complements rather than replaces CID because both technologies have some advantages: CID for smaller peptides with small charges, ETD for larger and multiply charged peptides (20, 21). An alternative way to utilize ETD is to use it in conjunction with CID because CID and ETD generate complementary sequence information (20, 22, 23). ETD-enabled instruments often support generating both CID and ETD spectra (CID/ETD pairs) for the same peptide. Although the CID/ETD pairs promise a great improvement in peptide identification, the full potential of such pairs has not been fully realized yet. In the case of de novo sequencing, de novo sequencing tools utilizing CID/ETD pairs indeed result in more accurate de novo peptide sequencing than traditional CID-based algorithms (23, 24, 25). However, in the case of database search, the argument that the use of CID/ETD pairs improves peptide identifications remains poorly substantiated. A few tools are developed to use CID/ETD (or CID/ECD) pairs for the database search but they are limited to preprocessing/postprocessing of the spectral data before or following running a traditional database search tool (26, 27). Nielsen et al., 2005 (22) pioneered the combined use of CID and ECD for the database search. Given a CID/ECD pair, they generated a combined spectrum comprised only of complementary pairs of peaks, and searched it with Mascot.⁵ However, this approach is hard to generalize to less accurate CID/ETD pairs generated by ion-trap instruments because there is a higher chance that the identified complementary pairs of peaks are spurious. More importantly, using traditional MS/MS tools (such as Mascot) for the database search of the combined spectrum is inappropriate, because they are not optimized for analyzing such combined spectra; a better approach would be to develop a new database search tool tailored for the combined spectrum. Recently, Molina et al., 2008 (26) studied database search of CID/ETD pairs using Spectrum Mill (Agilent Technologies, Santa Clara, CA) and came to a counterintuitive conclusion that using only CID spectra identifies 12% more unique peptides than using CID/ETD pairs. We believe that it is an acknowledgment of limitations of the traditional MS/MS database search tools for the analysis of multiple spectra generated from a single peptide.In this paper, we modify the generating function approach for interpreting CID/ETD pairs and further apply it to improve the database search with CID/ETD pairs. In contrast to previous approaches, our scoring is specially designed to interpret CID/ETD pairs and can be generalized to analyzing any type of multiple spectra generated from a single peptide. When CID/ETD pairs from trypsin digests are used, MS-GFDB identified 13% and 27% more peptides compared with the case when only CID spectra and only ETD spectra are used, respectively. The difference was even more prominent when CID/ETD pairs from Lys-N digests were used, with 41% and 33% improvement over CID only and ETD only, respectively.Assigning a p value to a PSM greatly helped researchers to evaluate the quality of peptide identifications. We now turn to the problem of assigning a p value to a peptide-spectrum-spectrum match (PS²M) when two spectra in PS²M are generated by different fragmentation technologies (e.g. ETD and CID). We argue that assigning statistical significance to a PS²M (or even PSⁿM) is a prerequisite for rigorous CID/ETD analyses. To our knowledge, MS-GFDB is the first tool to generate statistically rigorous p values of PSⁿMs.The MS-GFDB executable and source code is available at the website of Center for Computational Mass Spectrometry at UCSD (http://proteomics.ucsd.edu). It takes a set of spectra (CID, ETD, or CID/ETD pairs) and a protein database as an input and outputs peptide matches. If the input is a set of CID/ETD pairs, it outputs the best scoring peptide matches and their p values (1) using only CID spectra, (2) using only ETD spectra, and (3) using combined spectra of CID/ETD pairs.     

The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.