Scientists in the Robia lab work on a variety of projects focused on protein structure-function relationships and molecular physiology.  Major biological themes are summarized below, or check out our complete list of publications.

Regulatory Interactions of Transport ATPases

Protein-protein interactions of membrane proteins are difficult to quantify by traditional biochemical approaches such as coimmunoprecipitation or SPR.  These conventional methods require detergent solubilization, which can disrupt physiologically important interactions.  To circumvent this barrier we have developed methods for quantification of the affinity of transport ATPase regulatory interactions in the native environment of the membrane in living cells.  These FRET-based assays also reveal information about the quaternary structure of the protein complex.  We have focused on P-type transporters including SERCA1a, SERCA2a, and the sodium transporter NKA.  We have discovered novel determinants of functional modulation of these transporters by peptide regulators, and quantified how these regulatory interactions are tuned by phosphorylation by PKA/PKC.

1. Blackwell DJ, Zak TJ, Robia SL. Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer. Biophys J. 2016;111(6):1192-202. doi: 10.1016/j.bpj.2016.08.005. PubMed PMID: 27653478; PMCID: PMC5034344.
   
   
2. Soller KJ, Verardi R, Jing M, Abrol N, Yang J, Walsh N, Vostrikov VV, Robia SL, Bowser MT, Veglia G. Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides. Sci Rep. 2015;5:13000. doi: 10.1038/srep13000. PubMed PMID: 26292938; PMCID: PMC4543939.
   
   
3. Abrol N, Smolin N, Armanious G, Ceholski DK, Trieber CA, Young HS, Robia SL. Phospholamban C-terminal residues are critical determinants of the structure and function of the calcium ATPase regulatory complex. J Biol Chem. 2014;289(37):25855-66. doi: 10.1074/jbc.M114.562579. PubMed PMID: 25074938; PMCID: PMC4162186.
   
   
4. Ha KN, Masterson LR, Hou Z, Verardi R, Walsh N, Veglia G, Robia SL. Lethal Arg9Cys phospholamban mutation hinders Ca2+-ATPase regulation and phosphorylation by protein kinase A. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(7):2735-40. Epub 2011/02/02. doi: 10.1073/pnas.1013987108. PubMed PMID: 21282613; PMCID: 3041113.

Conformational Dynamics of Transport ATPases

A major focus of the Robia lab has been the structural basis of membrane transport.  Part of this effort has involved the development and utilization of “2-color transporter” constructs, novel sensors that report changes in ATPase conformation with altered intramolecular FRET.  They have applications for structural studies and are useful for high-throughput screening of drug libraries.  By combining these new tools with advanced spectroscopic methods and molecular dynamics simulations we have been able to reveal undiscovered enzymatic substates.  We have quantified microsecond/millisecond motions and identified discrete structural elements that are important for transporter function.  These important functional determinants are not accessible to traditional structural biology approaches such as X-ray crystallography.  Together with our collaborators, we have performed high throughput screening and discovered new small molecules that alter transporter structure and function.  This ongoing project seeks drug candidates that may be useful in the development of new therapies for human disease.  We have also implemented molecular dynamics simulations in our studies of ATPase conformational changes.  Comparison of the simulation results with data from physical experiments (fluorescence spectroscopy) allows us to interpret measurements made in cellular physiological environments in the context of high resolution structural information.

1. Iram SH, Gruber SJ, Raguimova ON, Thomas DD, Robia SL. ATP-Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening. Molecular pharmacology. 2015;88(1):84-94. Epub 2015/05/01. doi: 10.1124/mol.114.096792. PubMed PMID: 25924616.
   
   
2. Gruber SJ, Cornea RL, Li J, Peterson KC, Schaaf TM, Gillispie GD, Dahl R, Zsebo KM, Robia SL, Thomas DD. Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells. J Biomol Screen. 2014;19(2):215-22. Epub 2014/01/18. doi: 10.1177/1087057113510740 19/2/215 [pii]. PubMed PMID: 24436077. (cover article).
   
   
3. Hou Z, Hu Z, Blackwell DJ, Miller TD, Thomas DD, Robia SL. 2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding. PLoS One. 2012;7(7):e40369. Epub 2012/07/19. doi: 10.1371/journal.pone.0040369 PONE-D-12-07470 [pii]. PubMed PMID: 22808146; PMCID: 3394785.
   
   
4. Song Q, Pallikkuth S, Bossuyt J, Bers DM, Robia SL. Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase. J Biol Chem. 2011;286(11):9120-6. Epub 2011/01/12. doi: 10.1074/jbc.M110.198036 [pii]. PubMed PMID: 21220422; PMCID: 3059012.

Structural Mechanisms of Pathogenesis

The Robia lab has used fluorescence imaging and computational approaches for investigation of pathogenic mutations, revealing new information about the fundamental mechanisms of human diseases.  We and our collaborators have studied proteins relevant to diverse diseases including muscular dystrophy, vascular disease, heart disease, infectious disease, and cancer.