Correlating Amino Acid Sequence and Solvation to Disordered Protein Collapse Transitions and Phase Separation

Friday, September 14, 2018


Dr. Erik Martin

St. Jude Children's Research Hospital

Memphis, TN

erik martinHand 1144; 3:30 PM

Abstract: Cells actively maintain a vast array of complex, tightly regulated, chemical reactions, which occur on a multitude of time scales. The condensation of proteins, nucleic acids and other biomolecules by liquid-liquid phase separation (LLPS) into membraneless organelles likely plays an important role in organizing the myriad processes that sustain life. While this hypothesis has gained traction via macroscopic studies (ironically, using microscopy) of cellular organization, atomic-level understanding of the interactions that drive condensation of specific molecules and how these interactions influence emergent material properties of associated membraneless organelles remains elusive. Protein regions of low sequence complexity (LCRs) appear to be particularly well poised to serve as a model system for studying the requisite transient intermolecular interactions in atomic detail. A specific subset of LCRs with stereotyped amino acid compositions are particularly prone to demix at physiologically relevant solution conditions. These critical conditions for phase separation can be modulated by substituting amino acids with specific physicochemical properties, thus informing the thermodynamics of LLPS on a residue type basis. In order to characterize the driving forces and the molecular structure of associated systems underlying protein LLPS , experimental and computational techniques must feature the ability to resolve details on multiple length scales. Using a combination of small angle x-ray scattering (SAXS) and nuclear magnetic resonance (NMR) coupled to all atom Monte Carlo and coarse-grained simulations, it is possible to characterize both residue-specific interactions and the global properties of individual proteins and protein solutions. Combining insights from such multi-tiered experimental approaches make the goal of characterizing the structure of dense, yet dynamic, biomolecular assemblies at length scales approaching atomic detail within reach.

CV: 1997-2002 B.S.  University of Illinois, Biology Honors program.

2002-2010 PhD Biophysics & Computational Biology, University of Illinois, Urbana-Champaign.
Thesis Title: The Binding Pockets of QA and QB in the Photosynthetic Reaction Center of Rba. sphaeroides Probed by Pulsed EPR.

Current Research:
Postdoctoral Fellow, St Jude Children’s Research Hospital. 
Mentor: Dr. Tanja Mittag
Characterize physical and biochemical interactions of the yeast protein Ash1 with its cognate ubiquitin ligase.
• Develop techniques to prepare high quality samples from truncated regions of Ash1 protein and its binding partners using an array of protein biochemistry and molecular biology techniques. 
• Used unique combinations of multidimensional NMR analysis, including 2 and 3 dimensional carbon detected experiments, to assign all Ash1 backbone signals in spite of severe signal overlap. Using these assignments, mapped the specific, atomic level, interactions between Ash1 and the WD40 domain of the SCFCdc4 ubiquitin ligase.
• Applied BLI to determine thermodynamics of Ash1 binding.
• Developed all atom molecular dynamics simulations of Ash1 and quantified the shortcomings of current MD methods when simulating systems including disordered proteins.
Using the protein Ash1 as a model system, investigated the amino acid sequence determinates of the disordered protein conformational landscape upon multi-site phosphorylation.
• Used small angle X-ray scattering (SAXS) to determine the ensemble conformation of Ash1 and multi-site phosphorylated Ash1.
• Characterized multiple protein secondary structure propensities and cis / trans proline isomerization equilibrium using NMR spectroscopy.
• Used NMR relaxation measurements to discern minor differences between different phosphorylated species.
• Synthesized data from multiple experiments and Monte Carlo simulations.
Current Projects:
• Characterizing the interaction between Death Domain Associating Protein (DAXX) and the ubiquitin ligase substrate adapter MATH using NMR spectroscopy and exploring the conformational properties of DAXX.
• Using SAXS and NMR spectroscopy determining sequence dependence of the liquid-liquid demixing properties of the low complexity domain (LCD) of the RNA binding protein hnRNPA1.
Employment History:
2000 (December) Event Testing, Monsanto, St Louis, MO
2001   Field Researcher, Monsanto, Maui, HI
2003, 2007  Teaching Assistant, University of Illinois, Urbana-Champaign
    (Biophysics 401- Introduction to Biophysics)
2007, 2008  Teaching Assistant, University of Illinois, Urbana-Champaign
    (MCB 150- The Molecular and Cellular Basis of Life)
2010   Research Assistant, University of Illinois, Urbana-Champaign
2011 Postdoctoral Research Associate, University of Illinois, Urbana-Champaign
2012-present Postdoctoral Fellow, St Jude Children’s Research Hospital, Memphis, TN

Professional Organizations:

• American Association for the Advancement of Science.
• Biophysical Society.

Honors and Awards:

• “Teachers Ranked as Excellent by Their Students” - for teaching the course MCB 150 Spring 2007
• Life Science travel grant- 1 per department granted in 2009
• Principle Investigator on an NCSA/XSEDE supercomputing allocation – Fall 2013/Spring 2014
• Argonne National Lab Advanced Photon Source GUP #46417 – Active.
• Argonne National Lab Advanced Photon Source GUP #53593 – Active.
• Biophysical Society Intrinsically Disordered Proteins Subgroup Postdoctoral Award – BPS annual meeting 2017.

Research Interest:
My interests lie in using and developing diverse methodology to confront the challenges of characterizing intrinsically disordered proteins (IDPs) structurally and thermodynamically. A detailed understanding of sequence to conformational relationships in IDPs is essential in understanding myriad biological processes from signaling cascades to the formation of membraneless organelles.
Experimental Skills:
• Extensive, recent experience setting up and analyzing solution based two and three-dimensional NMR spectroscopy. 
• Designing and running pulsed EPR experiments, analyzing and simulating spectra. 
• Small Angle X-Ray Scattering (SAXS).  Experience includes organizing and constructing measurements, writing grants for beamline time and analyzing all data. 
• Synchrotron SAXS data collection and analysis.
• Designing binding experiments using Bio-layer interferometry (BLI – Octet systems) binding assays. 
• Extensive experience with multiple optical spectroscopic techniques including: fluorescence, FTIR, and kinetic visible light spectroscopy.
• All standard protein purification techniques as well as expression in E. coli (including isotopic labeling) and all accompanying biochemical and molecular biology techniques.
Computational and Bioinformatics Skills: 
• Expert in UNIX and the languages Matlab and TCL scripting. 
• Extensive scripting, custom curve fitting and large data set analyses using Matlab.
• Experienced with processing large data sets using Python, C and Awk languages. 
• Expertise with the molecular dynamics using the programs NAMD and Gromacs.
• Designing and running QM and QM/MM calculations using software packages such as Gaussian and PCGAMES. 
• Additional familiarity with small molecule docking software such as Glide and Haddock. 


Publications:

Rinyu, L., Martin, E.W., Takahashi, E., Maroti, P., and Wraight, C.A. 2003. Modulation of the free energy of the primary quinone acceptor (QA) in reaction centers from Rhodobacter sphaeroides: Contributions from the protein and protein-lipid (cardiolipin) interactions. Biochim. Biophys. Acta, 1655:93–101.

Martin, E. W., Samoilova, R. I., Narasimhula, K. V., Wraight, C. A., Dikanov, S. A. 2010. Hydrogen bonds between nitrogen donors and the semiquinone in the QB site of bacterial reaction centers. J. Am. Chem. Soc., 132:11671-11677.

Martin, E. W., Samoilova, R. I., Narasimhula, K. V., Tzu-Jen, L., O’Malley, P. J., Wraight, C. A., Dikanov, S. A. 2011. Hydrogen bonding to the semiquinones in the QA and QB sites of bacterial reaction centers. J. Am. Chem. Soc., 133:5525-5537.

Martin, E.W., Baldansuren, A., Lin, T.J., Samoilova, R.I., Wraight, C.A., Dikanov, S.A., 
O’Malley, P.J. 2012. Hydrogen bonding between the Q(B) site ubisemiquinone and Ser-L223 in the bacterial reaction center: a combined spectroscopic and computational perspective. J. Am. Chem. Soc,. 51:9086-9093.

Martin, E.W., Holehouse, A.S., Chrisy, G.R., Hughes, A., Pappu, R.V., Mittag, T., 2016. Sequence determinants of the conformational properties of an intrinsically disordered protein prior to and upon multi-site phosphorylation. J. Am. Chem Soc.  138:15323-15335.

Robertson, R.M., Yao, J., Gajewski, S., Kumar, G., Martin, E.W., Rock, CO., White, S.W., 2017. A two-helix motif positions the active site of lysophosphatidic acid acyltransferase for catalysis within the membrane bilayer, Nat. Struc. Mol. Biol., 24(8):666-671.

Wang, A., Conicella, A.E., Martin, E.W., Schmidt, H.B., Reeb, A.N., Ramirez Montero, D., Ryan, V.H., Rohatgi, R., Naik, M.T., Ayala, Y.M., Mittag, T., Fawzi, N.L., 2018. A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing, EMBO J., epub.

Martin, E.W., Mittag, T., 2018. The relationship of sequence and phase separation in protein low-complexity regions. Biochemistry, Article ASAP.

Bouchard, J., Otero, J., Scott, D., Szulc, E.M., Martin, E.W., Sabri, N., Marzahn, M.R., Peters, J., Salvatella, X., Schulman, B., Mittag, T., 2017. Cancer mutations of the tumor suppressor SPOP disturb the formation of enzymatically active, phase-separated compartments. Mol.Cell, accepted.

Martin, E.W., Peran, I., Holehouse, A.S., Pappu, R.V., Mittag, T., Sequence determinants of collapse in the hnRNPA1 low complexity domain., in preparation.



Talks:
“Sequence Determinants of the Conformational Properties of an Intrinsically
Disordered Protein Prior to and Upon Multi-Site Phosphorylation". Gordon Research Symposium – Intrinsically Disordered Proteins, Les Diablerets, Switzerland, 6/26/2016.

“Sequence Determinants of the Conformational Properties of an Intrinsically
Disordered Protein Prior to and Upon Multi-Site Phosphorylation". Biophysical Society – Intrinsically Disordered Protein Subgroup, New Orleans, LA 2/11/2017.

“Conformational properties integral to the phase separation properties of hnRNPA1 revealed by small angle X-ray scattering.” American Crystallographic Association, New Orleans, LA, 5/27/2017.

“The collapsed conformational landscape of the hnRNPA1 low complexity region revealed by SAXS, NMR and simulation.” CECAM Workshop, Paris, France, 10/5/2017.

“The collapsed conformational landscape of the hnRNPA1 low complexity region revealed by SAXS, NMR and simulation.” Biophysical Society – Intrinsically Disordered Proteins and Aggregates Platform, San Francisco, CA 02/20/2018.

“Hierarchical interactions on multiple length scales involving folded domains and disordered regions drive assembly and phase transitions.” Washington University, St Louis, MO, 03/05/2018.

“Correlating primary amino acid sequence and solvation to disordered protein collapse transitions and phase separation.” Bellairs Workshop on “The Physical Basis of Cellular Adaptation and Memory”, Barbados, 04/15/2018.
“The collapsed conformational landscape of the hnRNPA1 low complexity region revealed by SAXS, NMR and simulation.” Biophysical Society – Intrinsically Disordered Proteins and Aggregates Platform, San Francisco, CA 02/20/2018.

“Correlating primary amino acid sequence and solvation to disordered protein collapse transitions and phase separation.” GRS – Intrisically Disordered Proteins, Les Diablerets, Switzerland, 7/01/2018.

“Correlating primary amino acid sequence and solvation to disordered protein collapse transitions and phase separation.” University of Zurich, Zurich, Switzerland, 7/10/2018.


 


Poster presentations:
Martin, E. W., Wraight, C. A., Samoilova, R. I., Dikanov, S. A. 2009. ESEEM and HYSCORE analysis of QA- in native and 15N labeled reaction centers from Rhodobacter spharoides. Biophysical Society Annual Meeting.

Martin, E. W., Wraight, C. A., Samoilova, R. I., Dikanov, S. A. 2010.   Investigations of QA binding pocket mutations in Rhodobacter sphaeroides reaction centers using ESEEM and HYSCORE.  Biophysical Society Annual Meeting.

Martin, E. W., Wraight, C. A., Samoilova, R. I., Dikanov, S. A. 2010. Characterization of the secondary quinone (QB) binding pocket in photosynthetic reaction centers using pulsed EPR spectroscopy. Biophysical Society Annual Meeting.

Martin, E.W., Mittag, T., Lambert, L.J., 2013 Multiple phosphorylation sites in disordered regions of Ash1 form a dynamic complex with Cdc4. Biophysical Society Annual Meeting.

Martin, E.W., Mittag, T. 2014. The impact of molecular dynamics methods on the accuracy of simulations of the disordered protein Sic1.  Biophysical Society Annual Meeting.

Martin, E.W., Mittag, T. 2015. The dynamic complex between Cdc4 and Ash1 is facilitated by independent clusters of phosphorylated binding motifs.  Biophysical Society Annual Meeting.

Martin, E. W., Holehouse, A. S., Pappu, R. V., Mittag, T., 2016. Proline mediated conformational buffering in a multi-site phosphorylated protein. Biophysical Society Annual Meeting

Martin, E. W., Holehouse, A. S., Pappu, R. V., Mittag, T., 2016. Sequence determinants of the conformational properties of an intrinsically disordered protein prior to and upon multi-site phosphorylation. Gordon Research Conference - IDP. 

Martin, E. W., Holehouse, A. S., Pappu, R. V., Mittag, T., 2017. Sequence determinants of the conformational properties of an intrinsically disordered protein prior to and upon multi-site phosphorylation. Biophysical Society Annual Meeting.


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