@article{kaczowka_madding_epting_kelly_cianciolo_pizzo_2008, title={Probing the stability of native and activated forms of alpha(2)-macroglobulin}, volume={42}, ISSN={["0141-8130"]}, DOI={10.1016/j.ijbiomac.2007.09.019}, abstractNote={α2-Macroglobulin (α2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an ∼40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in α2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in α2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of α2M complexes.}, number={1}, journal={INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES}, author={Kaczowka, Steven J. and Madding, Lara S. and Epting, Kevin L. and Kelly, Robert M. and Cianciolo, George J. and Pizzo, Salvatore V.}, year={2008}, month={Jan}, pages={62–67} }
 @article{madding_michel_shockley_conners_epting_johnson_kelly_2007, title={Role of the beta 1 subunit in the function and stability of the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus}, volume={189}, ISSN={["0021-9193"]}, DOI={10.1128/JB.01382-06}, abstractNote={ABSTRACTThe hyperthermophilic archaeonPyrococcus furiosusgenome encodes three proteasome component proteins: one α protein (PF1571) and two β proteins (β1-PF1404 and β2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of theP. furiosusdynamic heat shock response (shift from 90 to 105°C) showed that the β1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the β2/β1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant α+β1+β2 CP assembled at 105°C was more thermostable than either the α+β1+β2 version assembled at 90°C or the α+β2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the α and β1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the β1 subunit in theP. furiosus20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that β subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.}, number={2}, journal={JOURNAL OF BACTERIOLOGY}, author={Madding, Lara S. and Michel, Joshua K. and Shockley, Keith R. and Conners, Shannon B. and Epting, Kevin L. and Johnson, Matthew R. and Kelly, Robert M.}, year={2007}, month={Jan}, pages={583–590} }