| Theodore
Feldman | Imaging
Compositional Changes in Mouse Bone at High Resolution THEODORE
C. FELDMAN, BHAVIN BUSA, and STEFAN JUDEX (SUNY Stony Brook, Stony Brook, NY 11794)
and LISA M. MILLER (National Synchrotron Light Source, Brookhaven National Laboratory,
Upton, NY 11973) Understanding
the changes in material properties of bone during formation is critical for diagnosing
and treating bone disease. Mechanical properties can be studied using assays such
as nanoindentation (NI), whereas synchrotron-based infrared microspectroscopy
(SIRMS) provides information on the chemical makeup of bone. In order to correlate
the mechanical and chemical properties, these two techniques must be performed
on the same sample. Typically, however, NI is performed on an embedded and polished
thick specimen, whereas SIRMS data are collected by measuring the transmission
of infrared light through thin, microtomed sections. In this work, we have developed
a new method for collecting SIRMS data in a reflection geometry, such that NI
and SIRMS can be performed on the same sample. This study validates the accuracy
and quality of reflection SIRMS, while investigating spatially-resolved chemical
changes in developing bone with respect to the sagittal axis. The tibia of two
male BALB/cByJ mice, 10 to 21 days of age, were dehydrated and embedded in poly-methyl-methacrylate
(PMMA). A 3 µm-thick sagittal section was cut from the surface of each embedded
bone. IR microspectroscopic imaging was performed on both the thin section and
the surface of the sample block in transmission and reflection geometry, respectively.
Transmission data were converted to absorbance using Beer's Law. Reflection data
were converted to absorbance through a Kramers-Kronig (KK) transformation. The
KK transformation will also be validated theoretically. The data from the two
imaging methods were compared using common visible landmarks present in both the
thin section and the embedded bone. Peak area ratios were performed on data from
both the thin section and sample block to provide quantitative measures of mineral
and matrix content and stoichiometry (i.e. phosphate-to-protein ratio; carbonate-to-phosphate
ratio). Data analysis indicates that there are disparities between transmission
and reflection data, particularly within the global phosphate-to-protein ratio.
Chemical indices of collagen cross-linking and the carbonate-to-phosphate ratio
appear consistent in trend and value across both time points and data collection
modes. Future studies will be necessary to determine the source(s) of these differences. |
| Katherine
Kao | Fluorescent
Studies of YiiP, a Membrane Transporter from E. coli.
KATHERINE
KAO (Stony Brook University, Stony Brook, NY 11794), DAXIONG FU, (Brookhaven National
Lab, Upton, NY 11973) & YINAN WEI (Brookhaven National Lab, Upton, NY 11973) Homeostasis
of Zinc ions in the cell is crucial to survival. YiiP is a metal transporter from
E. coli that plays a potential role in maintaining cellular Zn 2+ and Cd 2+ homeostasis.
Transport of these metals involves a coupled deprotonation mechanism and while
it is known that YiiP exists as a homodimer in the cell, its structure and mechanism
of transport has not yet been determined. In this study, a series of point mutations
W31F, W137F, W225C were made and fluorescent studies were performed on the mutants
to determine which tryptophan residue may be involved in metal binding. The mutants
were made by designing primers that contain the mutation and incorporating the
mutation by PCR reactions. To perform the fluorescent studies, His-YiiP and His-YiiPW31F
were over expressed using pET15b expression vector hosted in E. coli BL21cells.
The protein was prepared in membrane vesicles, solubilized and extracted from
vesicles by detergent (n-dodecyl ß-D maltopyranoside), purified by Ni-NTA
affinity column and followed by size exclusion chromatography. Fluorescent studies
were then performed and the natural fluorescence of tryptophan residues was excited
at 280nm. Emission spectra at 310-450 nm show that after Cd 2+ and ßMe were
added, the fluorescence intensity in YiiP significantly drops 77.1% indicating
that there is a significant conformational change that exposes more Trp residues
to a polar environment. When Cd 2+ and ß-Me were added to mutant W31F, the
fluorescence intensity dropped by 89.7%. This greater drop indicates that W31F
is not involved in mechanism of transport because a conformational change is still
induced when W31 is mutated. The greater decrease in fluorescence intensity is
due to the loss of a tryptophan residue by point mutation.
|
| Jessica
Newman | An
Antenna Design for the Mariachi Experiment
JESSICA
NEWMAN (Stony Brook University, Stony Brook, NY 11790), and HELIO TAKAI (Physics
Department, Brookhaven National Lab, Upton, NY 11793) This
component of the MARIACHI ((Mixed Apparatus Radar Investigation of Atmospheric
Cosmic-Rays of High Ionization) experiment focuses on the reception of reflected
radio signals from ionization clouds in the sky, which are largely caused by meteors
or cosmic rays. A new antenna model was acquired from the LOFAR experiment now
under construction in the Netherlands. The design is comprised of two inverted-V
dipoles. A dipole is a very important tool in radio reception. Although, its simplistic
design leaves a vacant gap directly overhead. This particular region is the most
active path of incoming cosmic rays that have been detected. Thus, the antenna
is blind to a crucial source of data. In order to compensate for this fact, two
dipoles are used. Each of which are oriented at 90 degrees to one another, so
that their fields of "view" overlap one another. As a result, this model
is commonly named a crossed dipole antenna. Using the crossed dipole model as
the foundation to the new design, an economical and functional antenna was built
with common materials such as standard PVC piping, aluminum tubing and wood. The
new antenna was relocated out doors where it replaced the previously commercial
unit. Prior to its placement outside, its range and basic features, such as current
and impedance, were calculated using a software-based Antenna Modeling Software
called EZNEC 4.0 and MultiNEC. Together these programs helped visualize the antenna's
scope of reception and the magnitude of the current flowing through the antenna.
Since this antenna detects as many occurrences as the last, its performance can
be rated adequate. Having an inexpensive and simple designed antenna will ensure
its ease for mass production. Thus, it will enable scientists to simultaneously
gather information from several parts of the sky. This would maximize results
and help determine the physical location of the source of these cosmic rays. Overall,
the goal of the MARIACHI experiment is to discover and learn about the origin
and cause of high energy cosmic rays. Efficient equipment appropriately arranged
for this type of detection will get us one step closer to finding where cosmic
rays come from. |
| Isaac
Pflaum | Exploration
of Conformational Transitions and Increased Sampling in Simulations of HIV Protease
ISAAC
PFLAUM1, DAVID STAMPF2 , JAMES DAVENPORT2and CARLOS SIMMERLING1,2,3
1 Department
of Chemistry, Stony Brook University
2 Computational Science Center, Brookhaven
National Laboratory
3 Center For Structural Biology, Stony Brook University
Catalytic
proteins, enzymes, are the biological machines responsible for life. Three dimensional
shapes and interactions are the root of enzyme activity . NMR and x-ray crystallography
provide time-averaged or ground state structures, which often do not explain a
protein's chemical activity. In contrast, simulation probes the dynamic behavior
of single molecules; however, computational resources restrict simulation time
scales to the order of nanoseconds. As a consequence, sampling of molecular conformations
is often insufficient to provide converged data , and molecular dynamics (MD)
based prediction of protein structure from sequence remains limited to relatively
small systems . Here I describe a method of increasing sampling during a MD trajectory
through Monte Carlo expansion. I have developed a new form of Concerted Rotation
, which employs Cyclic Coordinate Decent (CR-CCD), avoiding the instabilities
associated with similar techniques based on matrix inversion. CR-CCD features
large, coordinated movements of the protein backbone, irrespective of forces,
and adjusts the chi angles of side chains depending on the values of relevant
backbone dihedrals. My method uses a rotamer library to restrict the combinations
of backbone and side-chain angles to the distributions represented in samples
taken from the Protein Databank. This method is particularly useful for loop modeling,
where missing or poorly resolved segments of a protein are extrapolated using
only the sequence and chain closure constraints. The core of the method is based
on well understood theories of mechanics, which are often applied to actuated
linkage systems in robotics . It is through a fusion of work done in the dissimilar
fields of biochemical physics and robotics that we will make the best use of available
computer time, and speed the development of new drugs, enzymes and nanotechnologies.
My
efforts are directed towards implementing this method in AMBER , a MD simulation
package developed in an active collaboration of David Case at The Scripps Research
Institute, Tom Cheatham at the University of Utah, Tom Darden at NIEHS, Ken Merz
at Penn State, and my advisor, Carlos Simmerling, at SUNY-Stony Brook. AMBER development
is supported by the NSF, NIH, DOE and DARPA. The AMBER suite is widely used by
researchers to understand and predict the behavior of proteins, RNA, and DNA.
My development of the CR-CCD method was supported by fellowship grants from the
NSF, Battelle Foundation and the Center for Biotechnology at Stony Brook. I worked
in collaboration with faculty in the Departments of Chemistry, Applied Math and
Statistics and the Center for Structural Biology at Stony Brook and the Computational
Science Center at Brookhaven National Laboratory. As noted in the citations of
this proposal, in recent years, several concerted rotation methods have been developed
and applied to protein and RNA systems. To my knowledge, none have been integrated
into packages used as widely as the AMBER suite. This is a much needed application
that will benefit protein folding researchers for years to come. |
