Laboratory of Biophysics Brief overview I.Computational Biophysics Pastor, Venable, Skibinsky II.Mass spectroscopy & Protein Chemistry Boykins III.Spectroscopy.

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.