Determination of Dynein
Light Chain LC7 Stability and
Folding using Circular
Dichroism
Rachel Rasberry
HHMI Summer 2010
Working with Dr. Elisar Barbar
Background


Cytoplasmic dynein
 a large protein
complex
LC7
 a ubiquitous
component of
cytoplasmic dynein
 binds to Intermediate
Chain (IC)

http://www.nature.com/nrm/journal/v10/n12/box/nrm2804_BX1.html
Background

Cytoplasmic dynein
 is a motor protein
that converts
chemical energy
(ATP) into
mechanical energy
 transports cellular
cargo by “walking”
along microtubules
Relevance

Dynein plays a role in:
 Mitosis
 Vesicular
transport
 Development and maintenance of neurons
 Diseases



resulting from dynein dysfunction
Lissencephaly
Neural degeneration
Male infertility
LC7
a.
b.
Two
structural
views of
LC7
IC-LC7
complex
Barbar ’10 JOURNAL OF BIOLOGICAL CHEMISTRY
Bound IC
Completes the Fold
LC7 belongs to an ancient
protein superfamily.
The position of bound IC in the
IC-LC7 complex mimics a helix
that is integrated into the
primary structure in distantly
related LC7 homologs.
a.
b.
c.
IC-LC7 complex
MgI complex
MP1_p14 complex
Barbar ’10 JOURNAL OF BIOLOGICAL CHEMISTRY
Hypothesis

IC binding increases the stability of LC7.
Circular Dichroism (CD) Spectroscopy

CD

measures differences in the absorption of left-handed
polarized light versus right-handed polarized light
which arise due to structural asymmetry
 Can be used to:


determine whether a protein is folded, and if so characterize
its secondary structure
study the conformational stability of a protein under stress -thermal stability, pH stability, and stability to denaturants (i.e.
urea)
Determination of Protein Secondary
Structure by Circular Dichroism


Need to collect data in the
"far-UV" spectral region
(wavelengths of 190-250
nm)
Alpha-helix, beta-sheet,
and random coil
structures each give rise
to a characteristic spectral
profile of the CD spectrum
Determination of Protein Secondary
Structure by Circular Dichroism

An approximate fraction of each secondary
structure type that is present in any protein
can be determined

For example, CD can determine that a
protein contains about 50% alpha-helix;
however, it cannot determine where the
alpha-helical portions are located in the
molecule.
Experiment

LC7 concentrations tested
 30.0
μM
 16.7 μM
 9.0 μM
 6.0 μM
 3.3 μM
 1.4 μM
Far UV-CD spectra of LC7 wt (-)His6X Urea denaturation
Protein concentration: 3.3 μM
0.5 cm cell path length
30°C
Far UV-CD spectra of LC7 wt (-)His6X Urea denaturation
Protein concentration: 16.7 μM
0.1 cm cell path length
30°C
Urea unfolding profiles monitored by far-UV CD for LC7 wt (-)
His6X protein
30°C
protein concentrations:
 16.7 μM (monitored at
220 nm, cell path
length = 0.1 cm)
 3.3 μM (monitored at
222 nm, cell path
length = 0.5 cm)
Purifying the IC-LC7 complex
Residues 212-260 of IC (IC212-260) contain
the region of IC that binds to LC7
 Add SUMO protein and His-tag to IC212-260
 Run through nickel column and Size
Exclusion Column to purify

His tag
SUMO
IC
212
260
Purifying the IC-LC7 complex
Run a SDS-PAGE gel to check purity
 Mixed with LC7
 Run through a nickel column

 His-tag
allows binding to nickel
 Cut
with SUMO protease
 Elute IC-LC7 complex
His-tag_SUMO_IC212-260_LC7
Add SUMO
protease
Elute IC_LC7
His-tag_SUMO
Add 350μM
Imidazole
Continuation of project…
Perform CD experiments on the IC-LC7
complex
 Analyze data


Compare CD plots for free and bound LC7
Acknowledgements
Special thanks to:

Dr. Elisar Barbar
The Barbar Lab Group
 Jessica Morgan
 Yujuan Song
 Afua Nyarko
Dr. Kevin Ahern

Howard Hughes Medical Institute

