 My name is Christy Burriette. My name is Teron Yang. I'm Christy's graduate student in the Department of Chemistry at the University of Alabama at Birmingham. We're delighted that our article has been chosen to be featured in an upcoming issue of protein science. The title is, Membrane Protein Stability can be compromised by detergent interactions with the extra membranous soluble domains. We'll be joined by our co-office at Columbia, and together we'll describe some key points of our paper. My name is John Hunt. My name is Qi Wang. I'm currently a postdoc in John's lab in the Department of Biological Sciences at Columbia University. The current study grew out of efforts of less than more than a decade to express and characterize nucleotide-bite Nb1, or Nb1, which is an extra-membrane soluble domain for the cystic ribosus prance membrane conductance regulator. Nb1 is the site of the famous Delta F508 mutation, the most frequent mutation that causes the legal genetic disease cystic ribosus. The U.S. Cystic Ribosus Foundation sponsored a long-term project to purify Nb1 in functional form. It's turned out to be a heroic, biochemical, and biophysicist struggle. Eventually, in the two previously published papers cited by Christie, we demonstrated that the major obstacle to purification of Nb1 is its limited thermodynamic stability. These papers also show that the Delta F508 mutation lowers the stability of Nb1 further, and that this reduction is a major contributor to pathology caused by mutation. John and I thought studies on soluble Nb1 might help the consortium understand why some detergent extracted CFTR wasn't functional. These studies expand on our earlier NbD1 work, which was published in back-to-back papers in Protein Science in 2010. Here we studied 20 detergents, and to our surprise, some detergents that are generally considered to be mild actually denature NbD1. And furthermore, the relative harshness of the detergent mirrors the scale widely accepted for the interaction of detergents with the transmembrane domains, namely anionic and charged detergents are harsher than zwitterionic, which are harsher than nonionic. We conducted differential scanning calorimetry experiments on NbD1 in the presence of various detergents. The DSC measures the heat uptake of a protein sample during controlled heating. Here is an example of the DSC curves of NbD1 in the presence of increasing concentration of phospholene-14, a zwitterionic detergent. The midpoint of each endothermic peak Tmax represents the temperature at which 50% of the protein is unfolded and is widely used as a measure of the protein's thermostability. There is a gradual decrease in Tmax as the detergent concentration increases. At the highest detergent concentration, there is no more heat detected, indicating the protein is denatured by this detergent. To correlate the observed decrease in thermostability by DSC with possible structural change in the protein, John and Qi conducted circular dichroism experiments. Fire-UV circular dichroism spectroscopy, or CD, is an effective tool to monitor changes in protein secondary structure, and we use it to characterize such changes in NbD1 during thermal denaturation in the presence of detergents. To our surprise, several generally non-denaturing detergents induced a hypothetical non-native confirmation in NbD1 at temperatures where the domain is otherwise properly folded and stable as shown in the CD-titration data presented in the slide. Induction of this non-native structure coincided with the loss of the endothermic unfolding transition in NbD1 in the DSC experiment conducted by Wendy and Christie. These observations indicate that generally non-denaturing detergents can destabilize extracellular membrane soluble domains. This effect could represent a hidden obstacle to purification of internal membrane proteins in high yield for biochemical and structural studies.