Research Interests

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.

  1. Lamichhane R, Mukherjee S, Smolin N, Pauszek RF, 3rd, Bradley M, Sastri J, Robia SL, Millar D, Campbell EM. Dynamic conformational changes in the rhesus TRIM5alpha dimer dictate the potency of HIV-1 restriction. Virology. 2017;500:161-8. doi: 10.1016/j.virol.2016.10.003. PubMed PMID: 27821283; PMCID: PMC5327651.

  2. Dvornikov AV, Smolin N, Zhang M, Martin JL, Robia SL, de Tombe PP. Restrictive cardiomyopathy Troponin-I R145W mutation does not perturb myofilament length dependent activation in human cardiac sarcomeres. J Biol Chem. 2016. doi: 10.1074/jbc.M116.746172. PubMed PMID: 27557662.

  3. Abrol N, de Tombe PP, Robia SL. Acute inotropic and lusitropic effects of cardiomyopathic R9C mutation of phospholamban. J Biol Chem. 2015;290(11):7130-40. Epub 2015/01/17. doi: 10.1074/jbc.M114.630319. PubMed PMID: 25593317; PMCID: 4358133.

  4. Sastri J, Johnsen L, Smolin N, Imam S, Mukherjee S, Lukic Z, Brandariz-Nunez A, Robia SL, Diaz-Griffero F, Wiethoff C, Campbell EM. Restriction of HIV-1 by rhesus TRIM5alpha is governed by alpha helices in the Linker2 region. J Virol. 2014;88(16):8911-23. doi: 10.1128/JVI.01134-14. PubMed PMID: 24872590; PMCID: PMC4136267.

Oligomerization of Transmembrane Peptides

The activity of transport ATPases is often indirectly modulated through sequestration of regulatory partners into homooligomeric complexes.  We have used fluorescence microscopy and spectroscopy to quantify the affinity, stoichiometry, and quaternary conformation of these membrane protein oligomers.  We have developed a new method to quantify the rates of exchange of protomers from the oligomer. 

  1. Autry JM, Rubin JE, Pietrini SD, Winters DL, Robia SL, Thomas DD. Oligomeric interactions of sarcolipin and the Ca-ATPase. The Journal of biological chemistry. 2011;286(36):31697-706. Epub 2011/07/09. doi: 10.1074/jbc.M111.246843. PubMed PMID: 21737843; PubMed Central PMCID: PMC3173058.

  2. Zhao W, Waggoner JR, Zhang ZG, Lam CK, Han P, Qian J, Schroder PM, Mitton B, Kontrogianni-Konstantopoulos A, Robia SL, Kranias EG. The anti-apoptotic protein HAX-1 is a regulator of cardiac function. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(49):20776-81. Epub 2009/11/19. doi: 10.1073/pnas.0906998106. PubMed PMID: 19920172; PubMed Central PMCID: PMC2791603.

  3. Robia SL, Campbell KS, Kelly EM, Hou Z, Winters DL, Thomas DD. Forster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex. Circulation research. 2007;101(11):1123-9. Epub 2007/11/03. doi: 10.1161/CIRCRESAHA.107.159947. PubMed PMID: 17975108; PubMed Central PMCID: PMC2590498. (cover article)

  4. Robia SL, Flohr NC, Thomas DD. Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy. Biochemistry. 2005;44(11):4302-11. Epub 2005/03/16. doi: 10.1021/bi0478446. PubMed PMID: 15766259.

Biophysics of Protein Kinase Signaling

A key determinant of protein kinase specificity is the subcellular localization of the kinase near the target substrate.  Localization of kinases is a regulated process, and kinase activation is often accompanied by a translocation of the protein from one subcellular region to another.  We have used fluorescence microscopy observe this translocation and quantify important parameters such as the kinetics of binding/unbinding.  We have discovered novel structural elements that determine kinase targeting and rates of translocation.

  1. Koshman YE, Chu M, Engman SJ, Kim T, Iyengar R, Robia SL, Samarel AM. Focal adhesion kinase-related nonkinase inhibits vascular smooth muscle cell invasion by focal adhesion targeting, tyrosine 168 phosphorylation, and competition for p130(Cas) binding. Arteriosclerosis, thrombosis, and vascular biology. 2011;31(11):2432-40. Epub 2011/08/20. doi: 10.1161/ATVBAHA.111.235549. PubMed PMID: 21852560; PubMed Central PMCID: PMC3197899.

  2. Bossuyt J, Chang CW, Helmstadter K, Kunkel MT, Newton AC, Campbell KS, Martin JL, Bossuyt S, Robia SL, Bers DM. Spatiotemporally distinct protein kinase D activation in adult cardiomyocytes in response to phenylephrine and endothelin. The Journal of biological chemistry. 2011;286(38):33390-400. Epub 2011/07/29. doi: 10.1074/jbc.M111.246447. PubMed PMID: 21795686; PMCID: PMC3190922.

  3. Koshman YE, Kim T, Chu M, Engman SJ, Iyengar R, Robia SL, Samarel AM. FRNK inhibition of focal adhesion kinase-dependent signaling and migration in vascular smooth muscle cells.  Arterioscler Thromb Vasc Biol. 2010 Nov;30(11):2226-33. PubMed PMID: 20705914; PubMed Central PMCID: PMC4058887

  4. Robia SL, Ghanta J, Robu VG, Walker JW. Localization and kinetics of protein kinase C-epsilon anchoring in cardiac myocytes. Biophysical journal. 2001;80(5):2140-51. Epub 2001/04/28. doi: 10.1016/S0006-3495(01)76187-5. PubMed PMID: 11325717; PubMed Central PMCID: PMC1301406.