Determination of Dynein Light Chain LC7 Stability and

Folding using Circular Dichroism

Rachel Rasberry

HHMI Summer 2010Working 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. Two structural views of LC7

b. 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. IC-LC7 complex

b. MgI complex

c. 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 μM16.7 μM9.0 μM6.0 μM3.3 μM1.4 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

260212

SUMOHis tag IC

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 nickelCut with SUMO protease Elute IC-LC7 complex

His-tag_SUMO_IC212-260_LC7

Add SUMO protease

Elute IC_LC7

Add 350μM Imidazole

His-tag_SUMO

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