Axelrod, Herbert L.

Esonwune, Peace C.

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46a Sunday, February 21, 2010242-Pos
Comparative Study of the Effect of UV- VS. Gamma Radiation on Human
Hair
Ervin Palma1, David Gomez1, Eugene Galicia2, Yuri V. Griko3.
1San Jose State University, San Jose, CA, USA, 2Eloret Co., Sunnyvale, CA,
USA, 3NASA Ames Research Center, Mountain View, CA, USA.
The effect of UV- and 60Co gamma radiations on the structural and chemical
integrity of human hair has been studied to determine the feasibility of using
human hair as a non-invasive biomarker of radiation exposure to ionized and
non-ionized radiation. The goal was to identify the most sensitive molecular pa-
rameter associated with radiation-induced damage and to evaluate effects of
dose range from 0.5Gy to 10Gy. Steady state tryptophane fluorescence and
chemical analytical methods were utilized to quantitatively evaluate molecular
integrity of Trp fluorophor and SH-groups in hair proteins to asses the radiation
induced damage.
The study found that human hair fibers were progressively damaged by expo-
sure to both UV- and ionized gamma radiation up to 10Gy. Damage to the hair
was evidenced by a decrease in the fluorescence intensity due to depletion of
the amino acid tryptophan as well as significant reduction in a number of
free SH- groups.
The results show that hair-fibrils exposed to gamma rays of higher quantum en-
ergy than UV, undergo much smaller extent of changes in Trp fluorescence and
SH-group modification, then when exposed to equal energy of UV-irradiation.
The changes appear faster under the effect of UV than by gamma-irradiation
and their intensity and character depend on the dose. The stable Trp fluorophore
is extremely sensitive to UV-B and UV-C radiation in contrast to the ionized
gamma radiation, which causes damage from the reaction of free radicals
and direct deposition of energy.
We conclude that fluorescence intensity of the hair is a sensitive parameter for
the quantitative evaluation of the radiation exposure.
243-Pos
Direct Observation of Ostwald Ripening in Free-Interface Diffusion Based
Protein Crystallization
Aaron M. Streets1, Stephen R. Quake1,2.
1Stanford University, Stanford, CA, USA, 2Howard Hughes Medical
Institute, Chevy Chase, MD, USA.
In the late stages of first order phase transitions, interfacial energy minimization
drives competitive growth between precipitates of the new phase. This dissolu-
tion of small precipitates to feed the growth of larger clusters is commonly re-
ferred to as Ostwald ripening. Here, Ostwald ripening is directly observed dur-
ing the late stages of Lysozyme crystallization in a microfluidic free-interface
diffusion reactor. In order to measure the wide range of crystal sizes necessary
to quantitatively characterize the ripening phenomenon, we developed a novel
dynamic light scattering apparatus, with an incorporated microscope, based on
the microfluidic crystallization device. The crystal size distribution mean, ob-
served by scattering, grows as t 1/3 in agreement with the analytic predictions
of Lifshitz Wagner and Slyozov (LSW). In the case where a large crystal
emerges outside of the scattering volume, local crystal size distributions deviate
from power law growth in an expected fashion. A simulation based on LSW
theory is implemented to model the crystal size distribution evolution. The
observed growth trajectories show quantitative agreement with simulated
kinetics.
244-Pos
Conformational Flexibility of Aggregation-Prone Peptides Studied By
PET-FCS
So¨ren Doose1, Marc Lo¨llmann2, Michael Schwering3, Markus Sauer1.
1Julius-Maximilians University Wu¨rzburg, Wu¨rzburg, Germany, 2Bielefeld
University, Bielefeld, Germany, 3Ruprecht-Karls University Heidelberg,
Heidelberg, Germany.
Glycine-serine polypeptides in aqueous solution behave as unstructured poly-
mers that exhibit end-to-end contact rates dependent on contour length and
in accordance with polymer theory. Aggregation-prone peptides like polyglut-
amine (poly-Q, associated with various neurodegenerative diseases) of suffi-
cient length were shown to form more collapsed states in aqueous solution.
Conformational dynamics mediated by intrachain, interchain, and chain-
solvent interactions can reveal mechanistic aspects for the aggregation process
that is thought to be responsible for the onset of disease. We used contact-
induced fluorescence quenching due to photoinduced electron transfer (PET)
between terminally attached oxazine fluorophores and tryptophan for studying
peptide loop closure rates on ns-ms time scales. Conformational dynamics as
function of temperature, solvent viscosity, and osmolyte concentrations
were analyzed using fluorescence correlation spectroscopy (PET-FCS). Signif-
icantly smaller end-to-end contact rates for poly-Q and related peptides ascompared to poly-GS peptides with a viscosity-dependence hinting at internal
friction were found.
Membrane Protein Structure I
245-Pos
X-Ray Structure Determination of Isocytochrome C2 from the Photosyn-
thetic Bacterium Rhodobacter sphaeroides
Peace C. Esonwune1,2, Herbert L. Axelrod3.
1San Francisco State University, San Francisco, CA, USA, 2Stanford
University- Stanford Synchrotron Radiation Lightsource, Palo Alto, CA,
USA, 3Stanford University, Palo Alto, CA, USA.
In cytochrome c2-deficient strains of the photosynthetic bacterium, Rhodo-
bacter sphaeroides, a related c-type cytochome, isocytochome c2 replaces
cytochrome c2 as the secondary electron donor to the photo-oxidized bacterio-
chlorophyll dimer in the reaction center. We will describe the x-ray structure
determination of isocytochrome c2 at 2.2 A˚ from Rb sphaeroides.Analysis of
the x-ray diffraction data indicated the crystals belong to space group P3(2)
with unit cell dimensions a=b=56.0 A˚ c=90.5 A˚ and are merohederally twinned.
The structure of isocytochrome c2from Rb. sphaeroides shows significant struc-
tural similarity to the cytochrome c2 from a different photosynthetic bacterium,
Blastochloris viridis.
246-Pos
Structure Dynamics and Allosteric Regulation of the E. Coli High-Affinity
Methionine Transporter Metni
Eric Johnson, Chris Vercollone, Allen T. Lee, Douglas C. Rees.
Howard Hughes Medical Institute and Division of Chemistry and Chemical
Engineering, California Institute of Technology, Pasadena, CA, USA.
The high-affinity uptake of methionine by Escherichia coli is mediated by
MetNI, a member of the methionine uptake transporter family of ATP-binding
cassette (ABC) transporters. We previously reported the crystal structure of
MetNI at 3.7 A˚ resolution and a brief analysis of the methionine mediated
trans-inhibition of the transporters’ ATPase activity1. Here, we report two new
crystal structures of the MetNI transporter, solved at 2.8 and 4.0 A˚ resolution.
While both structures of MetNI reveal the transporter adopting an inward-
facing conformation, significant changes are observed in the conformation of
the MetI transmembrane, MetN nucleotide binding, andMetN-C2 carboxyl-ter-
minal regulatory domains. The conformational changes can be described
primarily as rigid-body movements which result in a partial closing of the
MetN nucleotide binding domains, and a simultaneous rotational rearrange-
ment of the MetN-C2 regulatory domain. The kinetic properties of trans-inhi-
bition have also been characterized by an analysis of ligand binding on the
ATPase activity of wild type and specific MetN-C2 regulatory domain mutants.
1. Kadaba N, Kaiser J, Johnson E, Lee A, and Rees D C. The high affinity
E. coli methionine ABC transporter: structure and allosteric regulation.
Science. 2008 Jul 11;321(5886):250-3.
247-Pos
Betp - X-Ray Structure and Function of An Osmosensor and Transporter
Reinhard Kraemer1, Susanne Ressl2, Vera Ott1, Sascha Nicklisch1,
Heinz-Juergen Steinhoff3, Lucy Forrest2, Christine Ziegler2.
1University of Koeln, Koeln, Germany, 2Max-Planck-Institute of Biophysics,
Frankfurt, Germany, 3University of Osnabru¨ck, Osnabrueck, Germany.
The Naþ coupled betaine uptake system BetP of Corynebacterium glutamicum
belongs to the BCCT family of transporters and comprises both a catalytic
function (betaine/Naþ cotransport) and a sensory/regulatory function respond-
ing to osmotic stress. Its 2D structure was recently solved by electron and its 3D
structure by X-ray crystallography. Within a trimeric structure, each BetP
monomer harbours both an N- and a C-terminal domain involved in stimulus
sensing and intramolecular signal transduction. Factors contributing to the sen-
sory and regulatory function of BetP are (i) the two terminal domains, (ii) Kþ
ions as an osmostress related stimulus, and (iii) interaction with the surrounding
membrane.
We used several techniques to analyze the contribution of the terminal sensory
domains to BetP function. By scanning mutagenesis we identified the signifi-
cance of single amino acids and parts of the C-terminal domain of BetP.
EPR spectroscopy was applied to determine the mobility or the C-terminal
domain under different functional conditions and to measure intra- and intermo-
lecular distances in BetP. The 3D structure of BetP finally revealed a putative
crosstalk between the three monomers of BetP via their C-terminal domains.
In addition, the observed asymmetry of the 2D crystal demonstrates the presence
of different conformational states of the three individual monomers.
On the basis of these studies we suggest a novel functional model of intersub-
unit crosstalk between the three individual monomers as well as the terminal
domains of BetP during its catalytic and its sensory function.


X-Ray Structure Determination of Isocytochrome C2 from the Photosynthetic Bacterium Rhodobacter sphaeroides 

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X-Ray Structure Determination of Isocytochrome C2 from the Photosynthetic Bacterium Rhodobacter sphaeroides

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