p53 R175H hydrophobic patch and H‐bond reorganization observed by MD simulation

Molecular dynamics simulations probe the origins of aberrant functionality of R175H p53, which normally prevent tumorigenesis. This hotspot mutation exhibits loss of its essential zinc cofactor, aggregation, and activation of gain of function promoters, characteristics contributing to the loss of normal p53 activity. This study provided molecular level insight into the reorganization of the hydrogen bonding network and the formation of a hydrophobic patch on the surface of the protein. The hydrogen bonding network globally redistributes at the expense of the stability of the β‐sandwich structure, and surface residues reorganize to expose a 250 Ų hydrophobic patch of residues covering approximately 2% of the solvent accessible surface. These changes could both stabilize the protein in the conformation exposing the patch to solvent to mediate the reported aggregation, and cause a destabilization in the area associated with DNA binding residues to affect the specificity. The development of the patch prior to loss of zinc indicates that stabilizing the patch quickly may prevent zinc loss. Considerations for rational design of small molecule therapeutics in light of the structural insight has been discussed and it suggest the positive ring around the hydrophobic patch and conserved residues may constitute a druggable site. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 176–185, 2016.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin™ as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin™ for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Electron spin relaxation measurements on the blue-copper protein plastocyanin: Deviations from a power law temperature dependence

Electron spin relaxation rates over the temperatue range 1.41-15.6 K are presented for the copper-containing protein plastocyanin. Measurements are described for two samples, each derived from a different preparation of equivalent purity, for which the ionic, redox, and protein compositions varied slightly. X-band data are analyzed in terms of a phonon-limited direct process and a Raman relaxation process, where the index of the Raman transport integral is treated as a fitting parameter. Both samples yield rate data at the highest temperatures that are characterized by small deviations from a simple Tⁿ power law dependence, with n in the range 4.8-5.2. These deviations are most easily quantified when the Tⁿ power law fits are compared with similar functions that allow for a finite cutoff in the phonon density of states corresponding to Debye temperatures between 90 and 100 K with n in the range 5.0-5.5.     

Kinetic evaluation, using 13N, reveals two assimilatory nitrate transport systems in Klebsiella pneumoniae.

A kinetic evaluation of initial rates of nitrate transport at concentrations between 1 microM and 1 mM indicated the presence of two transport processes. Analysis of the contribution of each process to the total activity permitted the determination of kinetic constants (Km) of 4.9 microM and 4.2 mM for the high-and low-affinity systems, respectively. The ratio of the maximal velocity of the high-affinity system to that of an apparent low-affinity system was about 0.3. Both systems were inhibited by the presence of NH4+ in the transport assay. Growth in the presence of equimolar NO3- and NH4+ repressed the synthesis of both systems when compared with growth in NO3- alone.     

Purification of an Endogenous polynucleotide phosphorylase from Brevibacterium JM98A.

Polynucleotide phosphorylase was purified form Brevibacterium JM98A (ATCC 29895). Homopolynucleotides were arsenolysed in the order polyadenylate greater than polycytidylic acid greater than polyuridylic acid greater than polyguanylate. The products were ribonucleoside 5'-monophosphates.