No abstract

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are receptor tyrosine kinases (RTKs) involved in the regulation of many important cellular processes. The current proposed models of activation are derived from structural studies using soluble extracellular domains and cytoplasmic tyrosine kinase domains. Preparations of full length IR and IGF1R have been hampered by the need for unconventional affinity chromatography resins and/or harsh eluting conditions. Here, we present a purification protocol to obtain full-length, detergent solubilized IR and IGF1R at quantities suitable for biochemical and structural characterization. We screened a panel of 24 structurally diverse detergents for optimal ligand activation. The receptors purified in n-dodecyl-β-D-maltoside showed ligand-stimulated autophosphorylation and kinase activity, suggesting an intact transmembrane signaling mechanism. This convenient purification protocol can be used to produce high quantities of IR, IGF1R, or other RTKs, and can be adapted for other challenging membrane proteins.

Fourier Transform Infrared Spectroscopy (FTIR) was used to quantify total monosaccharide content in the bacterium Enterobacter cloacae and several of its biofilm mutants. Bacterial biofilm samples were grown on trypticase soy agar, and 30 µL aliquots of aqueous sample bacterial plus biofilm were deposited into the center of barium fluoride crystals and dried at 50˚C for 1-hour before being scanned by FTIR. The total amounts of monosaccharides were estimated using the absorbance of the monosaccharide peak, 1192 -958 cm -1 , and normalized using the amide II peak, 1585 -1483 cm -1. This method provided a linear correlation between the absorbance of the monosaccharide peak and concentration of monosaccharide in standard monosaccharides, fructose, glucose, mannose, and rhamnose, over a concentration range of 0.5 -2.0 mg/mL.

Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in regulation DIM biosynthesis.