Biology subjects

Razavi, A.

Publications and source records attributed to Razavi, A..

4 recordsLinked to original sources

bioRxiv2024

P3 site-directed mutagenesis: An efficient method based on primer pairs with 3-prime overhangs

Site-directed mutagenesis is a fundamental tool indispensable for protein and plasmid engineering. An important technological question is how to achieve the efficiency at the ideal level of 100%. Based on complementary primer pairs, the QuickChange method has been widely used, but it requires significant improvements due to its low efficiency and frequent unwanted mutations. An alternative and innovative strategy is to utilize primer pairs with 3-prime overhangs, but this approach has not been fully developed. As the first step towards reaching the efficiency of 100%, we have optimized this approach systematically (such as use of newly designed short primers, test of different Pfu DNA polymerases and modification of PCR parameters) and evaluated the resulting method extensively with over 100 mutations on 12 mammalian expression vectors, ranging from 7.0-13.4 kb in size and encoding ten epigenetic regulators with links to cancer and neurodevelopmental disorders. We have also tested the new method with two expression vectors for the SARS-COV-2 spike protein. Compared to the QuickChange method, the success rate has increased substantially, with an average efficiency of [~]50%, with some at or close to 100 percent, and requiring much less time for engineering various mutations. Therefore, we have developed a new site-directed mutagenesis method for efficient and economical generation of various mutations. Notably, the method failed with a human KAT2B expression plasmid that possesses extremely GC-rich sequences. Thus, this study also sheds light on how to improve the method for developing ideal mutagenesis methods with the efficiency at [~]100% for a wide spectrum of plasmids.

molecular biology↗

bioRxiv2020

The Effects of Copper Addition on The Structure, and Antibacterial Properties of Biomedical Glasses

In this work, we studied the effects of copper incorporation in the composition of bioactive glass. Three different glass compositions were synthesized with 0, 3, and 6 mol% of copper addition. X-Ray Diffraction (XRD) patterns confirmed that an amorphous microstructure was obtained for all three glass compositions. Results from Differential Thermal Analysis (DTA) showed that the copper addition in the glass lowers the glass transition temperature, from 646{degrees}C to 590{degrees}C when added at 6 mol%. X-ray Photoelectron (XPS) survey and high-resolution scans were performed to study the structural effects of copper addition in the glass. Results indicated that the incorporation of copper changes the ratio of bridging to non-birding oxygens in the structure. Glasses were further analyzed for their structure with Nuclear Magnetic Resonance (NMR) spectroscopy, which indicated that copper acts as a network modifier in the glass composition and copper-containing glasses show a less connected microstructure. Antibacterial efficacy of the glasses was analyzed against E. coli and S. epidermis. Copper-containing glasses showed a significantly higher inhibition zone compared to control glass. The glass with 6 mol% copper, exhibited inhibition zones of 9 and 16mm against E. coli and S. epidermis bacteria, respectively.

bioengineering↗

bioRxiv2019

Reconciling simulated ensembles of apomyoglobin with experimental HDX data using Bayesian inference and multi-ensemble Markov State Models

Hydrogen/deuterium exchange (HDX) is a powerful technique to investigate protein conformational dynamics at amino acid resolution. Because HDX provides a measurement of solvent exposure of backbone hydrogens, ensemble-averaged over potentially slow kinetic processes, it has been challenging to use HDX protection factors to refine structural ensembles obtained from molecular dynamics simulations. This entails two dual challenges: (1) identifying structural observables that best correlate with backbone amide protection from exchange, and (2) restraining these observables in molecular simulations to model ensembles consistent with experimental measurements. Here, we make significant progress on both fronts. First, we describe an improved predictor of HDX protection factors from structural observables in simulated ensembles, parameterized from ultra-long molecular dynamics simulation trajectory data, with a Bayesian inference approach used to retain the full posterior distribution of model parameters. We next present a new method for obtaining simulated ensembles in agreement with experimental HDX protection factors, in which molecular simulations are performed at various temperatures and restraint biases, and used to construct multi-ensemble Markov State Models (MSMs). Finally, the BICePs algorithm (Bayesian Inference of Conformational Populations) is then used with our HDX protection factor predictor to infer which thermodynamic ensemble agrees best with experiment, and estimate populations of each conformational state in the MSM. To illustrate the approach, we use a combination of HDX protection factor restraints and chemical shift restraints to model the conformational ensemble of apomyoglobin at pH 6. The resulting ensemble agrees well with experiment, and gives insight into the all-atom structure of disordered helices F and H in the absence of heme. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/563320v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@187595dorg.highwire.dtl.DTLVardef@fbf9ddorg.highwire.dtl.DTLVardef@d13b72org.highwire.dtl.DTLVardef@95dba3_HPS_FORMAT_FIGEXP M_FIG Graphical TOC Entry C_FIG

biophysics↗

bioRxiv2017

How structural elements added in evolution from bacterial transporters serve human SLC6 homologs

Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters impart an expanded set of functional properties to the eukaryotic transporters, which are not shared by the bacterial homologs that lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters, that have been suggested to involve these structural elements, are sparse. To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov State Model analysis of experimentally determined properties of the wild type and mutant hDAT constructs, which reveal a rich spectrum of interactions of the hDAT N-terminus and the mechanisms by which these contribute to regulation (e.g., by phosphorylation), or to entirely new phenotypes (e.g., reverse uptake - efflux) added in evolution. We reveal separate roles for the distal and proximal segments of the much larger N-terminus shared by the eukaryotic transporters compared to the bacterial ones, consistent with the proposal that the size of this region increased during evolution to enable more, and different, modes of regulation that are not shared with the bacterial homologs.

biophysics↗