Michael Lewis

Structure and assembly of the endoplasmic reticulum: Biosynthesis and intracellular sorting of ERp61, ERp59, and ERp49, three protein components of murine endoplasmic reticulum

Rabbit antibodies have been prepared against ERp61, ERp59, and ERp49, three protein components of rough endoplasmic reticulum (RER) purified from mineral oil-induced plasmacytoma 315 (MOPC-315) tissue. Analysis of subcellular fractions of MOPC-315 tissue by an immunoprecipitation procedure demonstrated that all three endoplasmic reticulum proteins (ERps) were most enriched in the RER. Immunologically cross-reacting proteins of similar molecular weight have been detected in other eucaryotic cell lines. We have used these antibodies to study the post-translational processing and biosynthetic sorting of the three ERps in pulse-labeled MOPC-315 cells. No larger precursor forms of the ERps were detected and none of the ERps were found to possess asparagine-linked oligosaccharide moieties. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 cells to study the biosynthetic sorting of ERp61, ERp59 and ERp49 and have found no evidence to suggest that these proteins ever leave the endoplasmic reticulum. In addition, all three ERps appeared to have luminally exposed domains. ERp61 and ERp59 were entirely protected by the ER membrane in the absence of detergent, while ERp49 was a transmembrane protein that also possesses a cytoplasmically exposed domain. We have used the anti-ERp antibodies to quantitate the synthesis and accumulation of the three ERps during lipopolysaccharide (LPS)-induced lymphocyte differentiation. After 48 h of culture in the presence of LPS, the synthesis of ERp49 increased sixfold relative to that in control cells. The synthesis and membrane accumulation of ERp61 and ERp59 were less affected by the LPS treatment. Thus, membranes isolated from LPS-treated cells were enriched in ERp49 relative to those isolated from control cells.

Quantification of CH···π interactions: implications on how substituent effects influence aromatic interactions.

Attractive interactions between a substituted benzene ring and an α-substituted acetate group were determined experimentally by using the triptycene model system. The attractive interaction correlates well with the Hammett constants σ(m) (R(2)=0.90), but correlates much better with the acidity of the α-protons (R(2)=0.98).

The Use of Hammett Constants to Understand the Non-Covalent Binding of Aromatics

Non-covalent interactions of aromatics are important in a wide range of chemical and biological applications. The past two decades have seen numerous reports of arene-arene binding being understood in terms Hammett substituent constants, and similar analyses have recently been extended to cation-arene and anion-arene binding. It is not immediately clear why electrostatic Hammett parameters should work so well in predicting the binding for all three interactions, given that different intermolecular forces dominate each interaction. This review explores such anomalies, and summarizes how Hammett substituent constants have been employed to understand the non-covalent binding in arene-arene, cation-arene and anion-arene interactions.

Computational model for predicting experimental RNA and DNA nearest-neighbor free energy rankings.

Hydrogen-bonding, intrastrand base-stacking, and interstrand base-stacking energies were calculated for RNA and DNA dimers at the MP2(full)/6-311G** level of theory. Standard A-form RNA and B-form DNA geometries from average fiber diffraction data were employed for all base monomer and dimer geometries, and all dimer binding energies were obtained via single-point calculations. The effects of water solvation were considered using the PCM model. The resulting dimer binding energies were used to calculate the 10 unique RNA and 10 unique DNA computational nearest-neighbor energies, and the ranking of these computational nearest neighbor energies are in excellent agreement with the ranking of the experimental nearest-neighbor free energies. These results dispel the notion that average fiber diffraction geometries are insufficient for calculating RNA and DNA stacking energies.

A Computational Model for Predicting Experimental RNA Nearest-Neighbor Free Energy Rankings: Inosine•Uridine Pairs.

A computational model for predicting RNA nearest neighbor free energy rankings has been expanded to include the nonstandard nucleotide inosine. The model uses average fiber diffraction data and molecular dynamic simulations to generate input geometries for Quantum mechanic calculations. This resulted in calculated intrastrand stacking, interstrand stacking, and hydrogen bonding energies that were combined to give total binding energies. Total binding energies for RNA dimer duplexes containing inosine were ranked and compared to experimentally determined free energy ranks for RNA duplexes containing inosine. Statistical analysis showed significant agreement between the computationally determined ranks and the experimentally determined ranks.

Predicting the cation–π binding of substituted benzenes: energy decomposition calculations and the development of a cation–π substituent constant

This work proposes a new substituent constant, termed Π+, to describe cation–π binding using computational methods at the MP2(full)/6-311++G** level of theory with Symmetry Adapted Perturbation Theory (SAPT) calculations on selected cation–π complexes. The correlations between binding strength (Ebind or ΔH298) and common parameters for describing cation–π binding (∑σm, ∑σp, ∑(σm + σp), or Θzz) are decent (r2 between 0.79 and 0.90). SAPT calculations show that variations in the electrostatic (Eele), exchange (Eexch), induction (Eind), and dispersion (Edisp) component energies to the overall binding are almost entirely due to differences in arene–cation distances (dAr–cat). Eele varies most with dAr–cat; however, Eind seems to be the primary term responsible for the ∑σm, ∑σp, ∑(σm + σp) and Θzz parameters not accurately predicting the cation–π Ebind and ΔH298 values. The Π+ parameter largely reflects electrostatics, but it also includes the impact of exchange, induction, and dispersion on cation–π binding of aromatics, and the resulting correlation between ΔH298 or Ebind and Π+ is excellent (r2 of 0.97 and 0.98, respectively). Importantly, the Π+ parameter is general to cation–π systems other than those reported here, and to studies where the cation–π binding strength is determined using computational levels different from those employed in this study.

An enzyme-linked immunoassay for the detection of antibodies to endoplasmic reticulum☆

A simple, sensitive solid-phase assay for the detection of antibodies to endoplasmic reticulum is described. The assay is dependent upon the amount of antigen bound to the solid support and upon the amount of antibody bound to the support via the relevant antigen. The assay can be used to measure both polyclonal and monoclonal antibody to endoplasmic reticulum. It has been used to isolate several monoclonal antibodies which can recognize and precipitate specific proteins of the endoplasmic reticulum. In addition, it has been used to probe the membrane orientation of endoplasmic reticulum antigens.

The effects of varying the substituent and DNA sequence on the stability of 4-substituted DNA-naphthalimide complexes

DNA duplexes are stabilized by many interactions, one of which is stacking interactions between the nucleic acid bases. These interactions are useful for designing small molecules that bind to DNA. Naphthalimide intercalators have been shown to be valuable anti-cancer agents that stack between the DNA bases and exhibit stabilizing effects. There is a continued need to design intercalators that will exhibit these stabilizing effects while being more selective toward DNA binding. This work investigates 4-substituted naphthalimides with varying functional groups and their interactions with nucleic acid duplexes. Mode of binding was determined via wavelength scans, circular dichroism, and viscosity measurements. Optical melting experiments were used to measure the absorbance of the sample as a function of temperature. The Tm values derived from the DNA duplexes were subtracted from the Tm values derived from the DNA-intercalator complexes, resulting in ΔTm values. The ΔTm values demonstrated that the substituents on the intercalator affect the stability of the DNA-intercalator complex. From the results of this study and comparison to results from previous work, we conclude that the substituent type and position on the core intercalator molecule affect the stability of the complex it forms with DNA.