A Little Freedom Makes a Big Difference

As enzymes evolve over time, proteins of similar structure acquire small sequence changes and acquire new activities. What are the key changes in an enzyme’s structure or mechanism that allow this to happen? Researchers from the Hedstrom lab, led by former postdoc Gregory Patton, in collaboration with researchers from the Karolinska Institute, investigated this question in the case of two proteins, inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR). The enzymes share similar structural features but carry out different reactions in a cell. Since the two enzymes are in opposing pathways, there could be severe consequences if the enzymes slip and carry out the ‘other’ reaction.

The results, published last month in Nature Cell Biology, argue strongly that the difference is based on the ability of the enzyme to switch between two conformations. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR, including states in which the cofactor (NAD or NADP) is either in an ‘in’ conformation poised for hydride transfer (below, right), or an ‘out’ conformation in which the cofactor is 6 Å from IMP (below, left).

Using mutagenesis along with kinetic experiments, the group demonstrates that the ‘out’ conformation is required for the deamination of GMP. The accessibility of this conformation at the key step in GMPR but not IMPDH seems to determine the two different outcomes — thus, the freedom of the enzyme and cofactor to carry out a conformational change determines the specificity.

An interesting question, looking at the pathways, is whether GMPR can ‘run in reverse’, catalyzing the direct amination of IMP to form GMP (and saving the cell some energy in the process).  Overexpression of GMPR does allow E. coli to survive in the absence of IMPDH and GMPS, demonstrating that GMPR-driven synthesis of GMP can support life.  Indeed, some modern organisms that live in ammonia-rich environments appear to obtain GMP by this strategy.  If life began in an ammonia rich environment as is often proposed, the ancestral purine biosynthetic pathways may have produced GMP via GMPR.

For more details, see the paper:  Patton GC, Stenmark P, Gollapalli DR, Sevastik R, Kursula P, Flodin S, Schuler H, Swales CT, Eklund H, Himo F, Nordlund P, Hedstrom L. Cofactor mobility determines reaction outcome in the IMPDH and GMPR (beta-alpha)(8) barrel enzymes. Nat Chem Biol. 2011.

Long receives HHMI fellowship to develop new protein degradation strategy

Marcus Long, a 3rd yr PhD student in the Graduate Program in Biochemistry who works in the Hedstrom Lab, has been awarded a Howard Hughes International Student Predoctoral Research Fellowship for 2011-2013. This award, which is open only to students at selected universities, is given to roughly 40 international students in the life sciences per year in the US. The receipt of this award reflects strongly on the quality of research conducted in Brandeis, and particularly the interdisciplinary approach taken by principal investigator, Prof. Liz Hedstrom. Application for the award requires a clear research plan, which in this instance involves a novel protein degradation strategy (called IMPED), which was pioneered by Prof Hedstrom and her laboratory. Marcus will play his part in a collaborative effort  (alongside lab mates Rory Coffey, Devi Gollapali and established Hedstrom Group collaborators) to understand the mechanism and limitations of this new methodology.

Mehmet Fisek (BS/MS ’08), an alumnus of the Marder lab and undergraduate Neuroscience program at Brandeis, was also among the 48 winners named. Mehmet is currently doing graduate research in Rachel Wilson’s lab in the Dept. of Neurobiology at Harvard, working on olfactory neurophysiology in Drosophila.

See also story at Brandeis NOW.

Trimethoprim decorated beads for magnetically manipulating mammalian cells

Brandeis grad students Yue Pan (Chemistry) and Marcus Long (Biochemistry), together with postdoc Hsin-Chieh Lin and Professors Lizbeth Hedstrom and Bing Xu, have extended their previous work on 6 nm diameter magnetic nanobeads (comparable in size to a globular protein). They’ve shown that when decorated with the ligand trimethoprim, the nanobeads can be used to selectively bind to target E coli DHFR fusion proteins, and in addition can be used to manipulate live cells with a magnetic force. This work entitled “Cell Compatible Trimethoprim (TMP)-Decorated Iron Oxide Nanoparticles Bind Dihydrofolate Reductase (DHFR) for Magnetically Modulating Focal Adhesion of Mammalian Cells” is now online in the Journal of the American Chemical Society (JACS).

These small, magnetic beads are the first example of solid supported trimethoprim and have numerous advantages over larger traditional beads, including rapid purification, and ultra low non-specific binding. It is, however, their ability to affect live cells that is most important. In the paper they first show that Cos-1 and HeLa cells can be incubated with the beads for over 5 days with little cell death. These cells can subsequently be manipulated by transfection. Finally when exposed to a magnetic force, the focal adhesion of bead-treated Cos-1 cells can be manipulated.

See also: recommendation at Faculty of 1000

Steve Goldstein ’78 named provost

Steve A. N. Goldstein ’78, an undergraduate and postdoctoral alumnus of the Biochemistry department at Brandeis, has been appointed the next provost of Brandeis University.

see: story at The Justice ; story on BrandeisNOW ; Goldstein’s research website at U.Chicago.

Biochemistry Senior Research Talks on April 29

The Department of Biochemistry presents senior research talks by the 2010/2011 Biochemistry Honor and BS/MS Candidates on Friday, April 29, 11:30-1:30pm in Gerstenzang 121.

Benjamin D. Hornstein – BS/MS
Seq A: construction and analysis of mutants
Advisor: Sue Lovett

Marcus R. Kelly– BS/MS
Replacement Matrices for Transmembrane Proteins
Advisor: Douglas Theobald

Yuliya Y. Mints – BS/MS
Inosine Monophosphate Dehydrogenase and Transcription: a mechanism for retinitis pigmentosa?
Advisor: Liz Hedstrom

Sarah Naomi Olsen – BS/MS
Isolation, Purification, and Characterization of (+)-4R-limonene synthase
Advisor: Dan Oprian

Benjamin M. Whitlock – BS/MS
PABPN1 and SKIIP: A putative mechanism for the onset of Oculopharyngeal Muscular Dystrophy
Advisor: Dagmar Ringe

Philip D. Lessans – BS
Developing a Method of Extracting Native U snRNPs from eukaryotic cells using Snurportin 1 constructs
Advisor: Daniel Pomeranz Krummel

Jessica P. Liken – BS
Deletion Library Screen for Enhancers and Suppressors of ALS-associated FUS/TLS Toxicity in Yeast
Advisor: Greg Petsko

Everyone is welcome and encouraged to come. Pizza will be provided.

Strage Award Goes to Douglas Theobald

Prof. Gregory Petsko writes:

It is with great pleasure that I announce the recipient of the 12th Annual Alberta Gotthardt and Henry Strage Award for Aspiring Young Science Faculty, Dr. Douglas Theobald of the Biochemistry Department.

Doug is one of Brandeis’ most accomplished young faculty members. Since his arrival at Brandeis, he has consistently demonstrated the ability to think deeply about some of the most fundamental problems in biology. His work on the resurrection of ancient proteins is among the most exciting research in the field of molecular evolution. Using what he terms “paleocrystallography” — in reality, a sort of Jurassic Park from ancestral molecules — he is aiming to visualize the structural changes that occur during the evolution of enzymes and protein complexes. With the high-resolution structures of reconstructed ancestral molecules, correlated with functional data from biochemical analyses, Doug will be able to test experimentally specific evolutionary hypotheses about protein evolution and gain an understanding of what functions can be rationally engineered. […] A theoretician who also has both feet firmly grounded in experiments, Doug is also a gited teacher.

The award ceremony and lecture will take place on Monday, April 11 at 1:00 pm in Gerstenzang 121. The title of Prof. Theobald’s lecture will be “Evolution of structure and function in biological macromolecules”

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