cation, and fully cAMP saturated R subunits can be prepared by adding cAMP prior to the 
last gel-filtration step. The cAMP-free proteins were prepared as reported. Briefly, the R subunits were incubated with 5 M urea in 10 mM K-phosphate, pH 7.4, 50 mM KCl, 1 mM EGTA for 5 h at 4uC, followed by filtration through a prepacked PD-10 column and extensive buffer exchange using Amicon concentrators. Refolding of the proteins was carried out by extensive dialysis against the same buffer without urea and purification on a HiLoad Superdex 200 HR column. The absence of cAMP in the protein solution was checked by measuring complete occupancy of cAMP sites by c-AMP as reported. Circular dichroism CD spectra were recorded on a Jasco J-810 spectropolarimeter, equipped with a peltier element for temperature control. Experiments were performed in 10 mM K-phosphate buffer, 50 mM KCl, pH 7.4 using 2 mM of cAMP-free RIa forms, in the absence or the presence of 12755615 16632257 a large excess of cAMP. Spectra were acquired at the indicated temperature over the 195260 nm range at a scan rate of 50 nm/min. 4 scans were averaged for each spectrum. Buffer scans were recorded under the same conditions and subtracted. Thermal denaturation experiments were performed by monitoring changes in ellipticity at 222 nm at 1.5uC/min scan rate. Differential scanning calorimetry. Measurements were performed on a MicroCal VP-DSC differential scanning calorimeter with a cell volume of 0.527 ml at the indicated scan rate, customarily 1.5uC/min. Scans were performed over the 155uC range, unless indicated. Samples of RIa in its apo form were prepared in 10 mM K-phosphate buffer pH 7.4, 50 mM KCl, and centrifuged prior to measurements. cAMP was added at the indicated concentrations. Buffer-buffer baselines were acquired prior to the experiments and subtracted. Chemical baselines were removed prior to data fitting, using a cubic baseline routine. The subsequent excess molar heat capacity profiles ) were fitted to the following equation: DH:DH VH:K Cp~: 2: R T Materials and Methods Mutagenesis, expression and purification of RIa proteins DNA sequence corresponding to the human RIa, numbering according to Swiss-Prot Accession No. P10644, was amplified by PCR from the genomic DNA of human RIa and cloned into the pGEX-2T vector with a Factor Xa-cleavable Nterminal maltose binding protein-tag. Site directed AVE-8062 customer reviews mutagenesis of RIa to create G201E-RIa and G325D-RIa was performed using the QuickChangeTM kit. Full length human RIa was purified using a pGEX-KG/RIa construct as reported. Human RIa was expressed in E coli BL21 Codon Plus cells, induced at an OD600 of 0.60.7 with 1 mM IPTG, and grown for protein production for an additional 70 h at 30uC, when the bacteria were harvested. The pellets were resuspended in 100 ml of homogenization buffer and the bacteria broken by French press. The fusion protein was purified by affinity chromatography on amylose resin with elution by 1 M methyl-a-D-glucopyranoside and cleaved by incubation overnight at 4uC with Factor Xa , followed by gel- where DH is the calorimetric enthalpy, DHVH is the van’t Hoff enthalpy, R is the ideal gas constant, T is the absolute temperature and K is the equilibrium constant: DH VH: 1 1 { K~exp { T Tm R with Tm as the transition temperature. The theoretical DH based on crystal structures were obtained using well-known structure-energetics relationships as described previously. 2 March 2011 | Volume 6 | Issue 3 | e17602 Cross-b Aggregation
