laboratory of biophysics brief overview i.computational biophysics pastor, venable, skibinsky...
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Laboratory of Biophysics
Brief overview
I. Computational Biophysics Pastor, Venable, Skibinsky
II. Mass spectroscopy & Protein Chemistry Boykins
III. Spectroscopy Freedberg, Norris, Negin
IV. NMR Theory Bull
Characterization of:
proteins and peptides
carbohydrates and DNA
membranes and micelles
vaccines, blood & therapeutics
adjuvants & delivery systems
repeat units, Mening. B capsule polysaccharide
DPPC lipid bilayer
Human Hemoglobin
Characterization of:
proteins and peptides
carbohydrates and DNA
membranes and micelles
Using:
mass spec: Maldi-Tof, Quadrapole Ion Trap and Quadrapole-Tof
NMR: 300, 500 and 700 MHz
Light Scattering: multiangle laser LS interfaced with HPLC
Molecular Modeling: local cluster & graphics workstations
High-tech, Center-wide, expert users required , $$
Technique Physics Get Don’t get
Mass Spectrometry
separation of ionized
molecular fragments in
electric field
- mass of each fragment
- large proteins/mixtures
- atomic detail
- dynamics
Nuclear Magnetic
Resonance Spectroscopy
(NMR)
interaction of nuclear
magnets with each
other/magnetic field
- structure/composition
- conformation/dynamics
- large proteins
- details of averaging
model dependence
Light Scattering
Spectroscopy
angle/freq dependence
of scattered light
- mol wt, size, shape
- mixtures/aggregates
- atomic detail
- small molecules
Molecular Dynamics (MD)
computer simulation
Newtonian dynamics
of atoms in a molecular
assembly
detailed trajectory of
each atom in system
- long time/length
(>100 ns/100 Å)
Technique Physics Get Don’t get
Mass Spectrometry
separation of ionized
molecular fragments in
electric field
- mass of each fragment
- large proteins/mixtures
- atomic detail
- dynamics
Nuclear Magnetic
Resonance Spectroscopy
(NMR)
interaction of nuclear
magnets with each
other/magnetic field
- structure/composition
- conformation/dynamics
- large proteins
- details of averaging
model dependence
Light Scattering
Spectroscopy
angle/freq dependence
of scattered light
- mol wt, size, shape
- mixtures/aggregates
- atomic detail
- small molecules
Molecular Dynamics (MD)
computer simulation
Newtonian dynamics
of atoms in a molecular
assembly
detailed trajectory of
each atom in system
- long time/length
(>100 ns/100 Å)
Primary Review Responsibilities:
LAL test kits Pastor & Boykins
Adjuvants Bull, Pastor & Venable
Consultations/Support:
Protein/peptide chemistry Boykins
Mass spec Boykins
NMR Freedberg, Norris & Bull
Light Scattering Norris
Statistics of lot release Pastor & Bull
Computers/modeling Venable & Pastor
Four possibilities for product approval/lot release:
good product passes
good product fails
bad product fails
bad product passes
Four possibilities for product approval/lot release:
good product passes no problem
bad product fails no problem
Four possibilities for product approval/lot release:
good product fails problem for manufacturer
(manufacturer’s risk)
bad product passes problem for consumer
(consumer’s risk)Better characterization improves products/tests, reduces risks
Laboratory Presentations
Pastor:Computer Simulation of Membranes
Freedberg: NMR Spectroscopy of Carbohydrates
Norris: Light Scattering of Polysaccharides and Proteins
Bull: Theory and Experiment of NMR Relaxation
Venable: Computer Simulation of Carbohydrates and Proteins
Boykins: Mass Spectroscopy, Protein Chemistry
Origin of preservative effects of trehalose
Conformation of carbohydrates using Residual Dipolar Coupling
Meningococcal conjugate vaccines/Gates Foundation
Direct determination of H-bond in peptides/carbohydrates
Conformation of Mening. B capsule polysaccharide
Multiple peptide conjugates/malaria & anthrax vaccines
Exciting Result Since Site Visit
Collaboration with Laboratory of Biochemistry and Vascular Biology, Division of Hematology.
Problems with polymerized hemoglobin blood substitutes
Applied: mass spec, molecular modeling, light scattering oxidized raffinose cross-links- supposed to react with lys, - but also bind to 93cysnear heme pocket- prevents T-> R transition- accelerates iron release and heme degradationreduced functionality/toxicity
Exciting Result Since Site Visit
Collaboration with Laboratory of Biochemistry and Vascular Biology, Division of Hematology.
Problems with polymerized hemoglobin blood substitutes
Applied: mass spec, molecular modeling, light scattering “O-Raffinose cross-linked hemoglobin lacks site-specific
chemistry in the central cavity: structural and functional consequences of β93Cys modification”, Robert Boykins, Paul Buehler, Yiping Jia, Richard Venable, and Abdu Alayash Proteins: Structure, Function, and Bioinformatics (in press)
“Structural and functional characterization of glutaraldehyde polymerized bovine hemoglobin and its isolated fractions” Paul Buehler, Robert Boykins, Yiping Jia, Scott Norris, Darón
Freedberg and Abdu Alayash, submitted to Analytical Chemistry.
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