Mapping hydrogens in chymotrypsin structures with neutron diffraction

In a new paper “Time-of-flight neutron diffraction study of bovine γ-chymotrypsin at the Protein Crystallography Station” published in this month’s edition of the journal Acta Cryst F, Biochemistry grad student Louis Lazar and co-workers from the Petsko-Ringe lab report progress on their project to determine exact hydrogen positions in proteins using neutron diffraction.

Neutron diffraction was chosen, as opposed to X-ray diffraction, because one can visualize hydrogen species directly using neutrons, while it is extremely difficult and in most cases impossible to do so using X-ray diffraction. They chose the protein γ-chymotrypsin in order to determine hydrogen positions, as it fills the necessary requirements to be suitable for a neutron diffraction experiment. These requirements include a very large crystal size (> 1 mm3), moderately sized unit cell axes (no dimension greater than 100 Å), and it must be very stable as well as well-characterized. γ-chymotrypsin is the stereotypical serine protease, cleaving C-terminal to aliphatic and aromatic residues and containing a catalytic triad of serine, histidine, and aspartate. This information on hydrogen placement can then be applied to improve computational methods in which said placement is paramount, such as molecular modeling and rational drug design.

The paper details the collection of neutron data at pD (pH*) 7.1, with the help of the scientists at the Los Alamos National Laboratory. In particular, from the initial maps, they note that the catalytic histidine is doubly protonated, while the serine and aspartate making up the catalytic triad do not show density for the presence of deuterium. In order to complete the study of γ-chymotrypsin, data at a variety of pH values must be collected; data at pD (pH*) 5.6 has already been collected (Acta Cryst F65, 317-320), and data at pD (pH*) 9.0 will be collected in the future.

see also: full text of article (Brandeis users)

A molecular function of Zillion Different Screens protein explained

In a recent paper in Journal of Cell Biology entitled “Spatial regulation of Cdc55-PP2A by Zds1/Zds2 controls mitotic entry and mitotic exit in budding yeast“, Brandeis postdoctoral fellow Valentina Rossio and Assistant Professor of Biology Satoshi Yoshida reveal a molecular function of a mysterious protein Zds1.

The Zds1 protein in yeast  was identified some years ago in “a zillion different screens” for cell cycle mutants, stress response mutants, RNA metabolism mutants, etc., but the molecular function of the protein remained a mystery for more than 15 years. Rossio revealed that Zds1’s key target is a protein phosphatase 2A (PP2A) complex. She showed that Zds1 controls nucleocytoplasmic distribution of PP2A complex, and that this regulation is critical for cells to know when to enter and to exit from mitosis (picture below; cells lacking Zds proteins adopt an abnormal shape because of problems in mitosis). Rossio thinks all the other complicated phenotypes associated with ZDS1 can also be explained by PP2A regulation and is currently studying mechanistic details about the Zds1-PP2A interaction.

See also the accompanying commentary “Proteins keep Cdc55 in its place

Beckman Scholarships and URP Awards for Summer 2011

Beckman Scholars and Undergraduate Research Program Winners

Summer 2011

Beckman Scholars

The 2011 Beckman Scholars are:

Frank Scangarello (mentor: Suzanne Paradis, Biology)
Multivalent Metalloproteases Inhibitors to Increase Small Molecule Avidity and Selectivity to Study Semaphorin4D-Cleavage Mediated Synaptic Nerve Development

Zhequan Xu (mentor: Christine Thomas, Chemistry)
Novel Catalyst Design for Green Fuels

URP Recipients

(only students from the Division of Science are included in this list)

Heather Bernstein ’12 (Language & Linguistics; Neuroscience) with Prof. Stephen Van Hooser
Stimulus Therapy & its Implications for Rehabilitation: Using Channelrhodopsin-2 to determine spike time-dependent plasticity in neurons of the primary visual cortex in postnatal ferrets at eye opening

James En Wai Chin ’14 (Chemistry) with Prof. Lizbeth Hedstrom
IMP dehydrogenase nucleic acid association (How do IMPDH mutants affect IMPDH nucleic acid binding?)

Nimrod Deiss-Yehiely ’12 (Biology) with Prof. Sacha Nelson
A mouse model for Infantile Spasms involving TTX

Scott Finkelstein ’12 (Biology) with Prof. Paul Miller
Comparative Success of Strategies in a Continuous Iterated Prisoner’s Dilemma

Jessica Friedman ’13 (Biochemistry) with Prof. Tom Pochapsky
Insights into Substrate Recognition in Cytochrome P450cam

Julie Miller ’12 (Neuroscience) with Prof. Stephen Van Hooser
Roles of Inhibitory Neurons in Cortical Development

Anna Slavina ’12 (Psychology) with Prof. Art Wingfield
Selective syntactical attention among bilingual speakers

Sophie Travis ’13 (Biochemistry) with Prof. Dagmar Ringe
In vitro characterization of VPS35

Akash Vadalia ’12 (Biology; HSSP) with Prof. Angela Gutchess
Cross-Cultural Differences in the Specificity of Memory for Objects and Contexts

Alison White ’13 (Psychology) with Prof. Art Wingfield
Monitoring the Capacity of Short Term Memory

Abigail Zadina ’13 (Psychology) with Prof. Michael Rosbash
Huntington’s Disease: Insights into Mechanisms Involving Circadian Systems

Summer science classes at Brandeis

Enrollment for Summer School 2011 at Brandeis is open now (Session I starts on May 31). A number of science classes are being taught, from General Chemistry to Molecular Biotechnology. There is more information at the new Summer School Blog, including a story about Justin Dore, who is teaching a couple of the courses.

The Story Behind the Paper: How calmodulin became efficient

by John Lisman

Story behind: Nat Neurosci. 2011 Mar;14(3):301-4. Calmodulin as a direct detector of Ca2+ signals. Faas GC, Raghavachari S, Lisman JE, Mody I.

Long-term potentiation, a model for memory, is triggered by the activation of the calmodulin-dependent protein, CaMKII in dendritic spines. Sri Raghavachari, my former postdoc, and I were interested in how exactly CaMKII gets activated during LTP. It seemed that it should be straightforward to account for this in a computational model. Our confidence was based on the fact that a lot of groundwork had been done—the elevation of Ca2+ that triggered this process had been measured by Ryohei Yasuda and the interactions of Ca2+, calmodulin and CaMKII had all been determined in test tube experiments. But when Sri put this all together in a standard biochemical model, the simulations indicated that there would be virtually no CaMKII activation. Clearly something was wrong because Ryohei Yasuda had shown that under the same conditions in which he measured Ca2+ elevation in spines, he could also measure strong CaMKII activation.

During the summer I work at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and Sri came down to visit. We spent hours trying to figure out what was wrong with the simulations. Sri had carefully checked the simulations and determined that the program could accurately account for other data. Thus, the problem was not a bug in the program, but rather in an assumption we had put into the program. Every day, we awoke, convinced we had found the erroneous assumption; by nightfall we had rejected that idea.

One of the great things about working at MBL is the number of other neuroscientists there and the collegial atmosphere. We went to talk to Bill Ross, an expert in the measurement of Ca2+ in neurons. It had long been known that when Ca2+ enters neurons, very little of it stays free because most gets bound to “buffer” molecules. These are like the pH buffers all biochemists use; it’s just that Ca2+ buffers bind Ca2+ instead of protons. Bill asked us a lot about the particular Ca2+ buffers that were in spines—what was known about their molecular identity and their Ca2+ binding properties. Moreover, he wanted to know what assumptions about the buffers we had built into our simulations.

The answer to this was simple: we had followed the “standard” dogma based on the work of the Nobel Prize winner, Ernst Neher. He had determined that neurons contained a “fast buffer” that very rapidly binds the entering Ca2+. He had not been able to determine what type of molecule this was. Every model of Ca2+ dynamics that had since been developed had incorporated this fast buffer into the scheme and we had followed this convention. Thus, when Ca2+ entered the cytoplasm, 95% got bound to the fast buffer; only the remaining 5% was free and could activate calmodulin.

Perhaps there was something wrong with this assumption, but to evaluate this issue we had to learn about how Ca2+ buffers work, something we knew little about. Fortunately, Isabelle Llano was working as instructor in the MBL Neurobiology course. Because of her expertise in the small proteins that buffer Ca2+ in neurons, we went to chat with her. We learned a lot from her, but she also pointed us to Guido Faas, who was working with Istvan Mody at UCLA and measuring the kinetics of how Ca2+ binds to protein buffers. Previous work had measured the equilibrium properties of Ca2+ binding to proteins—the binding rate was then inferred by calculation. In contrast, Guido was using advanced methods to rapidly jump the Ca2+ concentration and then actually measure how fast Ca2+ would bind.

As we learned more about Ca2+ buffers from Guido and read the literature more carefully about calmodulin, we finally came up with a radical but intellectually satisfying new model—-perhaps the fast buffer that Neher had measured was none other than calmodulin itself. This would certainly radically change our computer simulations—-instead of calmodulin responding to only the 5% of Ca2+ that remained free after the bulk of Ca2+ was soaked up the unknown “fast buffer”, calmodulin would be activated by all the Ca2+ ions that entered, making the process of calmodulin activation and CaMKII activation much more efficient.

But for this to be true, calmodulin would have to bind to Ca2+ fast, faster than to the other major Ca2+ binding proteins in neurons (e.g. calbindin). When we talked to Guido about this possibility, he was excited to test it. His previous work had dealt only with calbindin, but he could now extend the work to calmodulin. Indeed, he could reconstitute the buffering in spines, putting both calmodulin and calbindin into his cuvettes. When the results came in, they were stunning; calmodulin has extraordinarily fast Ca2+ binding kinetics, much faster than that of calbindin.

With the new binding parameters provided by Guido, Sri reformulated his computer model. He took out the unknown fast buffer and replaced it with only calmodulin (which is at surprisingly high concentration) and calbindin. The simulations now showed that enough calmodulin was activated to account for the measured activation of CaMKII, our holy grail.

The new view we propose makes sense: Calmodulin is the transducer that couples Ca2+ entry to enzyme activation. It would make sense for calmodulin to be as efficient a detector of Ca2+ as possible and thus to directly intercept the entered Ca2+. Our results indicate that this is the case. Ca2+ triggered reactions are implicated in hundreds of forms of biological signaling. We therefore believe that this new view of Ca2+ signaling will have broad applicability.

2010-2011 Outstanding Teaching Fellows in Chemistry

Chemistry graduate students Mark Bezpalko, Xiachuan Cai and Fan Zhao will
be awarded Outstanding Teaching Fellow Awards this week for their excellent
work in general chemistry, organic chemistry, and advanced chemistry lab
sections, respectively. Their efforts were appreciated in many dimensions:

Mark was very effective, extremely reliable, and always well prepared and
patient with his students. During the lab he was very attentive, making sure
that his students were on the right track and showing a genuine interest in their
progress and development. He consistently did an excellent job evaluating
student work and providing advice and guidance to help them improve.

Xiaochuan had the highest numerical ratings of the graduate TFs in organic
chemistry and garnered such positive comments such as “Being very easy going
and always being ready to help a student in need” and “Very approachable and
knew the material to be covered”. Moreover, he was able to accomplish this while
still challenging his students and grading at the appropriate level.

Fan undertook the challenge to be the TF of a completely new lab course
focused on a frontier of chemistry—materials chemistry. He not only diligently
prepared each experiment, but also helped students with discussions of
background information and potential applications of the products targeted in
each experiment. He communicated well with the students, and the students
liked him very much.

MRSEC summer course in Optical Microscopy (June 20-24, 2011)

Optical microscopy has become a powerful experimental tool capable of simultaneously visualizing large scale structures such as entire cells, and fluorescently labeled single molecules within these complex structures. It has found important applications in diverse scientific fields.  The Brandeis Materials Science Research and Enginering Center will offer a one-week intense summer course in optical microscopy from June 20 – June 24, 2011, “Introduction to Optical Microscopy.“  The primary goal of the course is to train students in the fundamentals of microscopy and optics. The students will start by constructing a bright field and fluorescence microscope from simple optical components before learning how to use research grade optical microscopes. After completing the course, students will acquire knowledge necessary for using optical microscopes at limits of their capabilities and critically evaluating their performance.

This summer course is a condensed version of a popular graduate level course in  Quantitative Biology (Quantitative Biology Instrumentation Laboratory QB 120 b).  Our goal is to make this course accessible to students with all scientific backgrounds.  The course will be taught by Zvonimir Dogic, who is a faculty member in the Physics Department at Brandeis University.

More information and application procedures are available at the following website: http://www.brandeis.edu/mrsec/summercourses.html.

The Volen Center for Complex Systems Retreat, 2011

(co-written by Tilman Kispersky)

Introduction and Location

The annual Volen Center Retreat was held this week at the bucolic Warren Conference Center and Retreat in Ashland, Massachusetts.  The purpose of the one-day retreat is to provide a forum for conversation and encourage collaborations between members of the Brandeis and Volen center research communities.   Funded by the M.R. Bauer Foundation, the retreat features a distinguished invited speaker, lectures from Volen faculty that highlight the diversity of Neuroscience research at the Center and a poster session covering ongoing research projects of the members of the community.

The director of the Volen Center, Prof. Arthur Wingfield began the proceedings with a brief history of the retreat which is in its 17th consecutive year.  While historically the most common location for the retreat has been the Marine Biological Labs in Woods Hole, MA the retreat was held at the 220 acre property of the Warren Conference Center outside of Framingham this year.  Prof. Wingfield introduced the theme of the retreat: “Imaging: Recent breakthroughs in visualization – from synapses to circuits”.  Each lecture focused on data collected with advanced imaging techniques and highlighted how advanced optical methods had enabled a deeper understand of nervous system.

Lectures

The first lecture was given by Prof. Aniruddha Das from the Columbia University Department of Neuroscience.  Prof. Das’ research group developed a method to perform dual-wavelength imaging to measure both the volume of blood present in a given region of cortex as well as the oxygenation level of that blood, two quantities that are combined in traditional fMRI imaging.  Using dual-wavelength imaging Prof. Das found a task-related anticipatory haemodynamic signal in the visual cortex of awake monkeys.  This signal was unrelated to either single unit activity or any visual stimulation.  The finding suggests that cortical circuits increase their blood oxygenation level prior to the expected onset of a task in anticipation of the increased computational load.

The second speaker was Brandeis Professor Stephen Van Hooser.   Prof. Van Hooser studies motion detection in the visual system and is specifically interested in how motion selectivity develops and what role sensory inputs play in this process.  The ferret visual system, the animal model used by Prof. Van Hooser, develops orientation selectivity prior to receiving any sensory input.  However, motion selectivity requires visual inputs and thus develops later, after young ferrets open their eyes.  Prof. Van Hooser presented experimental results that employed two-photon imaging to simultaneously measure the activation of hundreds of cells at depths of up to 300 um beneath the cortical surface.  By presenting moving visual stimuli Prof. Van Hooser was able to track the emergence of motion selectivity in cortical neurons and was able to influence the course of development by changing the direction of motion of the stimulus.

Following the mid-day poster session, the afternoon portion of the retreat featured a trio of talks covering some of the cutting-edge imaging work currently being done at Brandeis.  First up was Dr. Avital Rodal (pictured at right), whose lab employs an innovative, high speed confocal microscopy technique to capture high-resolution images of tagged endosomes on the move in developing fly neurons.  By combining different markers in the same experiment, Dr. Rodal has been able to demonstrate transient interactions, undetectable by traditional methods.  Potentially, her work could help us understand a range of health issues in which endosomal trafficking has been implicated, including neurodegenerative disease and mental retardation.  See the moving endosomes for yourself in a recent blog post covering her exciting work!

The next speaker was able to remind us that sometimes it takes more than biologists to do biology — especially when the task is high-throughput image analysis.  Dr. Pengyu Hong, an Assistant Professor of Computer Science here at Brandeis, shared some of his work using High Content Screening, an automated method of analyzing image data and extracting information about cellular phenotypes and neurite length from images of cell cultures.  Using data provided by his collaborators around the world, his method is able to quantify neuronal morphology, allowing for high throughput genetic and drug discovery screening at improved levels of accuracy — a previously intractable task.

The final speaker of the retreat shared with us an intriguing work in progress.  Dr. David DeRosier (pictured at left), Brandeis Emeritus Professor of Biology, currently a member of the Turrigiano lab, has been developing an imaging technique called “Cryo-PALM”. If it sounds cool, it’s much more than that; it involves holding a biological sample frozen at no more than -140C, while imaging it with a room temperature microscope objective less than a millimeter away.  It sounds difficult — and as David told us, it is! — but the potential is huge.  Dr. DeRosier hopes to be able to precisely localize fluorescently labeled proteins in the synapse down to sub-nanometer resolution, and provide the most detailed picture ever of synaptic structure.

This year’s Volen center retreat was another success, with lots of informative talks, informal mingling, and even delicious food!

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