D. Meluzzi - Academia.edu (original) (raw)

Papers by D. Meluzzi

Lecture Notes in Computer Science, 2008

While nonribosomal peptides (NRPs) are of tremendous pharmacological importance, there is current... more While nonribosomal peptides (NRPs) are of tremendous pharmacological importance, there is currently no technology capable of highthroughput sequencing of NRPs. Difficulties in sequencing NRPs slow down the progress in elucidating the non-ribosomal genetic code and negatively affect various screening programs aimed at the discovery of natural compounds of medical importance. We propose to employ multistage mass-spectrometry (MS n ) for the data acquisition, followed by alignment-based heuristic algorithms for data analysis. Since mass spectrometry based analysis of NRPs is fast and inexpensive, this approach opens the possibility of high-throughput sequencing of many unknown NRPs accumulated in large screening programs.

RNA, 2012

Group I introns have been engineered into trans-splicing ribozymes capable of replacing the 39-te... more Group I introns have been engineered into trans-splicing ribozymes capable of replacing the 39-terminal portion of an external mRNA with their own 39-exon. Although this design makes trans-splicing ribozymes potentially useful for therapeutic application, their trans-splicing efficiency is usually too low for medical use. One factor that strongly influences trans-splicing efficiency is the position of the target splice site on the mRNA substrate. Viable splice sites are currently determined using a biochemical trans-tagging assay. Here, we propose a rapid and inexpensive alternative approach to identify efficient splice sites. This approach involves the computation of the binding free energies between ribozyme and mRNA substrate. We found that the computed binding free energies correlate well with the trans-splicing efficiency experimentally determined at 18 different splice sites on the mRNA of chloramphenicol acetyl transferase. In contrast, our results from the trans-tagging assay correlate less well with measured trans-splicing efficiency. The computed free energy components suggest that splice site efficiency depends on the following secondary structure rearrangements: hybridization of the ribozyme's internal guide sequence (IGS) with mRNA substrate (most important), unfolding of substrate proximal to the splice site, and release of the IGS from the 39-exon (least important). The proposed computational approach can also be extended to fulfill additional design requirements of efficient trans-splicing ribozymes, such as the optimization of 39-exon and extended guide sequences.

Physical Review Letters, 2006

The extraordinary complexity of Lennard-Jones (LJ) clusters, which exhibit numerous structures an... more The extraordinary complexity of Lennard-Jones (LJ) clusters, which exhibit numerous structures and &amp;quot;phases&amp;quot; when their size or temperature is varied, presents a great challenge for accurate numerical simulations, even without accounting for quantum effects. To study the latter, we utilize the variational Gaussian wave packet method in conjunction with the exchange Monte Carlo sampling technique. We show that the quantum nature of neon clusters has a substantial effect on their size-temperature &amp;quot;phase diagrams,&amp;quot; particularly the critical parameters of certain structural transformations. We also give a numerical confirmation that none of the nonicosahedral structures observed for some classical LJ clusters are favorable in the quantum case.

Nucleic Acids Research, 2013

The 3D higher order organization of chromatin within the nucleus of eukaryotic cells has so far r... more The 3D higher order organization of chromatin within the nucleus of eukaryotic cells has so far remained elusive. A wealth of relevant information, however, is increasingly becoming available from chromosome conformation capture (3C) and related experimental techniques, which measure the probabilities of contact between large numbers of genomic sites in fixed cells. Such contact probabilities (CPs) can in principle be used to deduce the 3D spatial organization of chromatin. Here, we propose a computational method to recover an ensemble of chromatin conformations consistent with a set of given CPs. Compared with existing alternatives, this method does not require conversion of CPs to mean spatial distances. Instead, we estimate CPs by simulating a physically realistic, bead-chain polymer model of the 30-nm chromatin fiber. We then use an approach from adaptive filter theory to iteratively adjust the parameters of this polymer model until the estimated CPs match the given CPs. We have validated this method against reference data sets obtained from simulations of test systems with up to 45 beads and 4 loops. With additional testing against experiments and with further algorithmic refinements, our approach could become a valuable tool for researchers examining the higher order organization of chromatin.

Nature Chemical Biology, 2007

Polyketides are clinically important natural products that often require elaborate organic synthe... more Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.

Bioorganic & Medicinal Chemistry Letters, 2008

Mass spectrometry (MS) is an important tool for studying non-ribosomal peptide, polyketide, and f... more Mass spectrometry (MS) is an important tool for studying non-ribosomal peptide, polyketide, and fatty acid biosynthesis. Here we describe a new approach using multi-stage tandem MS on a common ion trap instrument to obtain high-resolution measurements of the masses of substrates and intermediates bound to phosphopantetheinylated (holo) carrier proteins. In particular, we report the chemical formulas of twelve diagnostic MS 3 fragments of the phosphopantetheine moiety ejected from holo carrier proteins during MS 2 . We demonstrate our method by observing the formation of holo-AcpC, a putative acyl carrier protein from Streptococcus agalactiae.

Bioorganic & Medicinal Chemistry, 2011

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete '... more Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-bketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.

Annual Review of Biophysics, 2010

Knots appear in a wide variety of biophysical systems, ranging from biopolymers, such as DNA and ... more Knots appear in a wide variety of biophysical systems, ranging from biopolymers, such as DNA and proteins, to macroscopic objects, such as umbilical cords and catheters. Although significant advancements have been made in the mathematical theory of knots and some progress has been made in the statistical mechanics of knots in idealized chains, the mechanisms and dynamics of knotting in biophysical systems remain far from fully understood. We report on recent progress in the biophysics of knotting-the formation, characterization, and dynamics of knots in various biophysical contexts.

The estimation of contact probabilities (CP) from conformations of simulated bead-chain polymer m... more The estimation of contact probabilities (CP) from conformations of simulated bead-chain polymer models is a key step in methods that aim to elucidate the spatial organization of chromatin from analysis of experimentally determined contacts between different genomic loci. Although CPs can be estimated simply by counting contacts between beads in a sample of simulated chain conformations, reliable estimation of small CPs through this approach requires a large number of conformations, which can be computationally expensive to obtain. Here we describe an alternative computational method for estimating relatively small CPs without requiring large samples of chain conformations. In particular, we estimate the CPs from functional approximations to the cumulative distribution function (cdf) of the inter-bead distance for each pair of beads. These cdf approximations are obtained by fitting the extended generalized lambda distribution (EGLD) to inter-bead distances determined from a sample of chain conformations, which are in turn generated by Monte Carlo simulations. We find that CPs estimated from fitted EGLD cdfs are significantly more accurate than CPs estimated using contact counts from samples of limited size, and are more precise with all sample sizes, permitting as much as a tenfold reduction in conformation sample size for chains of 200 beads and samples smaller than 10 5 conformations. This method of CP estimation thus has potential to accelerate computational efforts to elucidate the spatial organization of chromatin.