ANL Biosciences Division

1
ANL Biosciences Division

• Carol S. Giometti, Acting Division Director
• www.bio.anl.gov

2
Where BIO Fits into ANL
3
Funding Portfolio
4
Why Does DOE Fund Biological Research?

• Post-WWII interest in biological effects of
  radiation
• Current DOE Science Mission Drivers, all related
  to Energy Production
• Bioremediation of DOE Waste Sites
• Climate Change
• GenomicsGtL
• Biofuels
• Bioremediation
• Carbon sequestration
• Health effects of low-dose radiation

5
DOE-Funded BIO Research at ANL
6
Biogeochemistry to Explore Natural Processes
Green rust forms naturally in suboxic
environments, the product of both abiotic and
microbially induced corrosion of iron as well as
microbially mediated and abiotic reductive
dissolution of ferric oxyhydroxides by Fe2.
TEM image of Green Rust particle (large hexagonal
particle) that has reduced U(VI) to U(IV) in the
form of uraninite nanoparticles (small black dots
on the edges of GR particle). (E. OLoughlin)
7
Microbial Processes Relevant to Bioremediation

• Minerals commonly found in soils, together with
  the bacteria and the extracellular material
  associated with them, are thought to play a key
  role in determining an element's speciation and
  thus its mobility in the subsurface.
• X-ray fluorescence mapping of single bacterial
  cells of the metal-reducing bacteria Shewanella
  oneidensis

(K.Kemner, S. Kelly)
8
Identification of Microbial Outer Membrane
Proteins
9
Neutravidin and Silver-stained 2D gels of
Biotinylated Surface Proteins
(C. Giometti)
10
Informatics/bioinformatics
Comparative proteome analysis (2DE/MS),
integrated with genome sequence data, provides
the capability to deduce metabolic and regulatory
mechanisms.
(G. Babnigg)
11
Study of cytochromes from Geobacter
sulfurreducens, bacteria important for
bioremediation
X-ray
diffraction
12-heme cytochrome Protein nano-wire?
Novel swapped PAS sensors discovered
Periplasmic sensor domain from GSU0935
Overlap of two 3-heme c7 structures
(M. Schiffer)
12
High-throughput Protein Expression and
Purification of DOE Protein Targets
GenomicsGTL

• Production capacity 400-800 targets/week
• Targets currently include proteins involved in
  cellulose degradation
• Cytochromes involved in electron transport
  relevant to metal reduction in bioremediation
  also over expressed
• Proteins of unknown function are key targets

(F. Collart)
13
Climate Change
Controls on soil carbon retention under elevated
atmospheric CO2

• Specific questions guiding research
• How long will soil C accrual induced by CO2
  enrichment continue?
• What is the potential longevity of the accrued C?
  
• Does atmospheric CO2 enrichment cause fundamental
  changes in the cycling of C inputs into and
  through various SOM pools?
• How does changing the relative apportionment of C
  sources (root vs. leaf litter) affect SOM
  dynamics?
• How do species-specific responses and edaphic
  properties influence the potential for soil C
  accrual?

• Meta-analysis of 35 independent experimental
  observations supports generality of results and
  suggests CO2 enrichment might increase soil C by
  5.6 over a decade (median accrual rate of
  0.19 Mg C ha y-1)
• Critical mechanisms include
• Species-specific root turnover and magnitude of
  the below-ground productivity response to CO2
  enrichment
• Presence of unsaturated soil mechanisms for
  protecting C inputs from rapid decomposition

(Julie Jastrow)
Jastrow et al., 2005, Global Change Biology
112057-2064 Matamala et al., 2003, Science
3021385-1387
14
Restoration of Tallgrass Prairie at Fermilab
Fermilab was established in 1969 in Batavia,
Illinois. The site had been farmland for over 100
years, with corn, wheat and dairy activities.
Restoration of native tallgrass prairie began in
1974. Annual plantings create a chronosequence of
restorations encompassing over 400
hectares. Potential approach to carbon
sequestration.
(M. Miller)
15
Structural Biology Center
19ID and 19BM beamlines
(A. Joachimiak)
16
NIH Funded Research
17
Membrane Protein Production in Rhodobacter
Advantage of the Rhodobacter expression system
This organism can be engineered to provide
coordinated synthesis of foreign membrane
proteins with synthesis of new membrane into
which they can be incorporated.
Invaginations of the cell membrane found in
species of Rhodobacter
(D. Hanson/P. Laible)
18
Over Expression of Membrane Proteins
DK Hanson PD Laible
Genes representing entire membrane proteomes are
being cloned into the Rhodobacter membrane
protein expression system with 80 efficiency.
Ligation-independent cloning enabled a
significantly higher-throughput approach to the
test for successful heterologous expression for
this target set.

• 400 Rhodobacter expression constructs have been
  evaluated.
• Overall Rhodobacter expression success is 60.

(D. Hanson/P. Laible)
19
Structural Genomics (MCSG)
(A. Joachimiak)
20
GM/CA CAT
National Institute of General Medical Sciences
and National Cancer Institute Collaborative
Access Team
Complete Sector Three Beamlines and Laboratory
Office Module
B. Fischetti)
21
Human Antibody Engineering for Ultrastabilization
Protein engineering is being used to produce
ultrastable antibody molecules for use as
scaffolds for protein chips and other
applications. (Fred Stevens)
(F. Stevens)
22
Stem Cells What Triggers Differentiation?
2D-electrophoresis analysis of membrane proteins
extracted from hES cells
hES cells expressed cell-surface antigens
specific for undifferentiated hES cells
L. Chen/Q. Jin
23
New Initiative Projects (LDRD)
24
Enzymes for cellulosic ethanol production
structure-function studies
Enzymes critical for lignocellulose degradation

• 5 enzyme domains studied during FY2007
• 3 crystallized and X-ray diffraction data
  collected at SBC
• structure determination efforts are underway
• Collaboration with scientists at USDA, Peoria, IL

• Acetyl esterase from Trichoderma reesei
• 326 amino acids
• 2.9 Å X-ray data collected

• Glucuronoyl esterase from Ruminococcus
  flavefaciens 17
• organism present in cow rumen
• enzyme from cellulosome
• 418 aminoacids
• 1.9 Å X-ray data collected

• Glucuronoyl esterase from Trichoderma reesei
• 375 aminoacids
• 1.8 Å X-ray data collected

(R. Pokkuluri)
LDRD Project started FY2007
25
Single Cell Detection Hybrid phage (virus)
imaging/therapeutic probes
Remove DNA
Fusion of affinity reagent via phage display
Replace with functional core
Energy Dispersive X-ray Analysis
Cobalt phages
Magnetic moment for each cobalt phage 4.7 x 104
mB
(L. Chen)
26
Genes Involved in Cadmium-induced bone loss
Cd stimulates bone loss within 8-24 h of exposure
in mice In 2-4 h, Cd increases expression of
genes involved in (I) protection of bone cells,
including metallothioneins 12 and transferrin
receptor and (II) formation and activation of
osteoclasts Results support hypothesis that Cd
increases bone demineralization via p38MAPK
pathway that leads to stimulation of
osteoclast-mediated bone resorption
Osteoclast (multinucleated involved in bone
resorption)
Osteoblasts (involved in bone formation)
(M. Bhattacharrya)
27
Angiogenesis
Vascular development, both normal and aberrant,
is critically dependent upon the activities of
endothelial cells (ECs). These cells, which line
the lumen of all the blood vessels of the body,
are the ringmaster of a process called
angiogenesis - the generation of new capillary
blood vessels.
Gene expression studies are revealing how this
process is regulated.
28
Metalloproteins - Identification of Metal-binding
Proteins Critical to Life, Disease and
Bioremediation

• Transient interactions of proteins with metals is
  a layer of rapid, post-translational regulation
  not assessed by conventional techniques.
• Goal is to develop methods using the APS that
  will provide systematic identification of metal
  binding proteins.

A simultaneously run gel which was subsequently
stained is shown at identical proportions to the
XRF scan. The total amount of protein loaded onto
the lane was 40 ?g of hemoglobin which, with a
molecular weight of 64,000 and 4 iron atoms per
tetramer, corresponds to approximately 2.5 ng or
45 pmoles of iron.
29
Molecular Recognition and Prediction of Small
Molecule Binding to Proteins Using Small and Wide
Angle Scattering

• Goals
• developing a novel approach to genome-wide
  identification of small molecule binding proteins
  
• Develop software to compare large numbers of
  ligand-binding peptides to whole genome sequences
• 
  
  (L. Makowski)

Effect of increasing concentration of guanidine
hydrochloride on the solution scattering profile
from hemoglobin. The black curve is 0 M guanidine
hydrochloride, the red curve is 2M guanidine
hydrochloride and the blue curve is 4M guanidine
hydrochloride.
30
Other Initiatives

• Ricketts Center
• Institute for Genomics and Systems Biology
• Metagenomics

31
Advanced Protein Crystallization Facility