Columbus Labs

Linda Columbus

Linda — Mon, 18 Feb 2008 22:33:35 +0000

PI

Postdoctoral Research: The Scripps Research Institute
Advisors: Kurt Wuthrich and Scott Lesley
NMR and X-ray crystallography of Membrane proteins.

PhD: The University of California Los Angeles (2001)
Advisor: Wayne Hubbell
Mapping protein backbone dynamics with EPR Spectroscopy and site – directed spin labeling.

BA, high honors: Smith College (1996)
Advisor: David Bickar

Professor Columbus is interested in accelerating membrane protein techniques to investigate the structure and dynamics of membrane proteins involved in bacterial pathogenesis.

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Linda’s CV

Identification and removal of nitroxide spin label contaminant: Impact on PRE studies of α-helical membrane proteins in detergent

Linda — Sat, 17 Mar 2012 18:40:04 +0000

Kroncke B.M. and Columbus L. Protein Science 21: 589–595 (2012).

NMR paramagnetic relaxation enhancement (PRE) provides long-range distance constraints (∼15–25 Å) that can be critical to determining overall protein topology, especially where long-range NOE information is unavailable such as in the case of larger proteins that require deuteration. However, several challenges currently limit the use of NMR PRE for α-helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label to the protein-detergent complex that can result in spurious PRE derived distance restraints. The effect of the nitroxide spin label contaminant is evaluated and quantified and a robust method for the removal of the contaminant is provided to advance the application of PRE restraints to membrane protein NMR structure determination.

Alison Dewald successfully defended her thesis

Linda — Wed, 18 Apr 2012 15:44:10 +0000

On Friday April 13th, 2012, Alison Dewald presented her thesis “Lipid reconstitution and functional characterization of outer membrane Opacity-associated (Opa) proteins of Neisseria”. Her private defense followed and her committee approved her PhD degree with minor revisions to her written document. The lab and Alison’s thesis committee celebrated with champagne afterwards.

Alison answers questions after her public presentation.

Time to celebrate!

The committee and lab toast to Alison's success.

The Dewald family.

Lab and family celebration for alison and Brett.

Tsiga Solomon

Linda — Sun, 19 Apr 2009 19:26:27 +0000

Laboratory Specialist

BS: Chemistry, University of Virginia (2012)

Tsiga’s research aims to further elucidate nixtroxide spin label dynamics at solvent exposed sites on α-helical membrane proteins. This project follows on the heels of a paper published by the lab (Biochemistry 49:10045–10060 (2010)) in which a model for nitroxide motion in an aliphatic environment was proposed. Tsiga will be extending this project to encompass a greater diversity of sites on α-helical membrane proteins using multiple nitroxide side chains, EPR linewidth simulations and X-ray crystallography on a model system α-helical membrane protein, LeuT.

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

Linda — Wed, 04 May 2011 02:14:01 +0000

Dewald AH, Hodges JC, Columbus L. Biophysical Journal 100:2131 – 2140.
The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)₆₀ and Opa₅₀ into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.

Ryan Lo presents his 3rd year poster.

Linda — Wed, 18 Apr 2012 15:54:13 +0000

On April 12th, the third year chemistry graduate students presented their research at the annual poster event. Ryan Lo presented his poster entitled “Effects of detergent, ionic strength, and temperature on the quality of beta-barrel membrane protein NMR spectra”

Ryan Lo presents his 3rd year poster.

Alison Dewald

Linda — Wed, 17 Sep 2008 21:34:58 +0000

Postdoctoral Fellow

PhD: Chemistry, University of Virginia (2012)
BS: Chemistry, Pennsylvania State University (2001)
M. Ed: Temple University, Educational Psychology (2006)
Alison conducted undergraduate research in the Guiltinan lab at the Penn State Biotechnology Institute, then taught chemistry and biology in Hershey, PA.

Alison’s research uses liposome (lipid vesicle) systems to investigate membrane proteins involved in bacterial pathogen – host interactions. Ultimately, her research aims to utilize the cellular hijacking mechanisms invented by pathogens (e.g. inducing phagocytosis, controlling cellular trafficking, and causing cell death) in the design of protein-decorated liposomes with novel characteristics desirable for drug delivery.

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Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites

Linda — Sun, 24 Oct 2010 20:20:33 +0000

Kroncke BM, Horanyi PS, and Columbus L. Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites. Biochemistry 49:10045–10060 (2010)

Brett Kroncke successfully defended his thesis

Linda — Wed, 18 Apr 2012 15:19:21 +0000

On Wednesday April 11th, 2012, Brett Kroncke presented his thesis “Nitroxide Spin Label Motion on Helical Membrane Proteins”. His private defense followed and his committee approved his PhD degree with minor revisions to his written document. The lab and Brett’s thesis committee celebrated with champagne afterwards.

Brett presents his thesis research.

Let the celebrations begin!

The committee and the lab toast to Brett.

Dan Fox gives Brett a present (a Marshall original of the Rotunda) from the lab.

Brett socializes with the committee.

He doesn't look any different?

Brett Kroncke

Linda — Tue, 19 Feb 2008 14:24:05 +0000

Postdoctoral Fellow

PhD: Chemistry, University of Virginia (2012)
BS: Chemistry, University of Wisconsin-Madison (2007)
Brett worked with Dr. John Wright on ultrafast pulse recognition hardware and software design and implementation.

Brett aims to use quantitative information obtained from EPR spectra to improve structure and dynamics determination for α-helical membrane proteins. His work investigating the structure and dynamics of spin labels on a model membrane protein, LeuT, has provided a quantitative understanding of the EPR lineshape of detergent/lipid exposed nitroxides. Currently he is working with a model protein, TM0026, to assess the degree to which this understanding can improve paramagnetic relaxation enhancement (PRE) derived nuclear magnetic resonance (NMR) α-helical membrane protein structure determination.

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Structure of the GLD-1 homodimerization domain: insights into STAR protein-mediated translational regulation.

Linda — Sun, 04 Apr 2010 20:27:44 +0000

Beuck C, Szymczyna BR, Kerkow DE, Carmel AB, Columbus L, Stanfield RL, Williamson JR. Structure of the GLD-1 homodimerization domain: insights into STAR protein-mediated translational regulation. Structure 18:377-89 (2010).

Dan and Ryan honored at the 2012 Robert J. Huskey Research Exhibition

Linda — Wed, 28 Mar 2012 02:14:10 +0000

The Huskey Research Exhibition is an annual event which provides a forum for all graduate students in the Graduate School of Arts and Sciences and the School of Architecture to publicize their research. In addition, the event rewards students presenting superior research. The 12th Annual Huskey Research Exhibition occurred on March 21, 2012. This year, Dan Fox and Ryan Oliver received second place in the Physical Science and Mathematics Division. Ryan’s poster was titled “Investigating the relationship between physical properties of detergents and membrane protein structure determination” and Dan’s poster was entitled “NMR solution structure of Opa₆₀: a Neisserial membrane protein that mediates host phagocytosis”.

Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

Linda — Sat, 09 May 2009 05:13:03 +0000

Columbus L, Lipfert J, Jambunathan K, Fox DA, Sim AYL, Doniach S, and Lesley SA. Mixing and Matching Detergents for Membrane Protein NMR Structure Determination J. Am. Chem. Soc. 131:7320–7326 (2009)

Ashton Brock

Linda — Sat, 19 Nov 2011 22:03:12 +0000

Graduate Student

BS: Chemistry, Winthrop University (2011)
Ashton conducted undergraduate research with Dr. Takita Sumter on the regulation of High Mobility Group A1 proteins involved in colon cancer.

Ashton’s research aims to determine suitable detergents that solubilize and support the function of lipoprotein signal peptidase A, a membrane bound protease. She aims to determine which properties of the selected detergents enable function and why that property is required for enzymatic activity.

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Kroncke and Columbus paper highlighted in Protein Science

Linda — Sun, 18 Mar 2012 17:32:09 +0000

Identification and removal of nitroxide spin label contaminant: Impact on PRE studies of α-helical membrane proteins in detergent published this month in Protein Science was highlighted “In This Issue” by the journal.