Characterization of Dynein Solubility
using Small Ubiquitin-like Modifiers.
Brian Phan
Dr. Elisar Barbar
HHMI
Summer 2009
Introduction
X
•pH levels
•Solvent
•Temperature
•Concentration
•Time
Microtubule
ATP
Converts chemical energy from
ATP into mechanical energy.
Transports cellular cargo
along microtubules.
Cellular Cargo
Relevance
 Structural and mechanistic dysfunction in dynein
can provide understanding to diseases and
disorders.
 Provide insight into producing pharmaceutical
agents that prevent aggregation.
 Leads into further studies for protein chemistry.
Background (cont.)
Small ubiquitin-like modifier (SUMO).
Protein modifier which attaches itself to other
protein substrates.
Discovered in 1996.
Helps protein regulate cellular processes.
http://www.mskcc.org/mskcc/_assets/content-image/215901.jpg
Hypothesis: SUMO helps the dynein intermediate
chain become more soluble in solution.
Methods
 Grow and purify SUMO-protease.
 Use SUMO protease to cleave off SUMO from ICdel
 Use gel to identify if enzyme is active in cleaving off
SUMO
Results
MW
1
55kDa
43kDa
34kDa
26kDa
2
3
4
5
SDS Gel of SUMOprotease after
Affinity
Affinity
Buffer Content:
purification.
20mM Na-Phosphate
1. Molecular
Marker
500mM
NaCl
5mM
2. B-mercaptoethanol
Flowthrough
5% Glycerol
3. Wash
10mM Imidazole
4. Na-azide
50mM Imidazole
1mM
5.
17kDa
350mM Imidazole
µV
histSUMOprotease_072409_BP_2.DATA
Affinity Buffer Content:
STH 10.00
20mM Na-Phosphate
500mM NaCl
5mM B-mercaptoethanol
5% Glycerol
10mM Imidazole
1mM Na-azide
pH=9.0
SPW 0.20
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
-200
-400
-600
-800
-1,000
-1,200
-1,400
-1,600
-1,800
-2,000
-2,200
-2,400
-2,600
-2,800
RT [min]
0
5
10
15
20
25
30
35
40
45
50
 SEC Chromatogram for SUMO protease where Abs280 nm is
plotted against elution time on 45 mL SEC Column. Buffer used
was common affinity buffer.
10
µV
histSUMOprotease_073009_BP_1.DATA
0
-10
-20
-50
-60
STH 10.00
-40
SPW 0.20
-30
-70
-80
-90
-100
-110
-120
-130
-140
-150
-160
-170
-180
-190
RT [min]
0
10
20
30
40
50
60
70
80
90
 SEC Chromatogram for SUMO protease where Abs280 nm is
plotted against elution time on 90 mL SEC Column.
µV
1,400
1,300
histSUMOprotease_080309_BP_1.DATA
Aff. Buffer Content:
1,200
1,100
50mM Tris-Base
350mM NaCl
10mM Imidazole
1mM B-mercaptoethanol
20% sucrose
1,000
900
800
700
500
400
STH 10.00
600
pH=8.0
200
100
SPW 0.20
300
0
-100
RT [min]
0
10
20
30
40
50
60
70
80
90
 SEC Chromatogram for SUMO protease where Abs280 nm is
plotted against elution time on 90 mL SEC Column. Buffer used
was prescribed by Structural Biology Program at Cornell
University.
µV
280
260
Aff. Buffer Content:
240
220
200
180
120
STH 10.00
160
140
histSUMOprotease_081109_BP_1.DATA
50mM Tris-Base
350mM NaCl
10mM Imidazole
1mM B-mercaptoethanol
No sucrose
80
60
40
SPW 0.20
100
pH=9.0
20
0
-20
-40
RT [min]
-60
0
20
40
60
80
100
120
140
 SEC Chromatogram for SUMO protease where Abs280 nm is plotted
against elution time on 90mL SEC Column. Buffer used was prescribed
by Structural Biology Program at Cornell University.
µV
histSUMOprotease_081209_BP_1.DATA
STH 10.00
Affinity Buffer Content:
20mM Na-Phosphate
500mM NaCl
5% Glycerol
10mM Imidazole
1mM Na-azide
pH=9.0
SPW 0.20
360
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
-20
-40
-60
-80
RT [min]
0
10
20
30
40
50
60
70
80
90
100
 SEC Chromatogram for SUMO protease where Abs280 nm is
plotted against elution time on 90 mL SEC Column. Buffer used
was original buffer.
Results
MW
43kDa
1 hr at 30oC
SUMO-CAT: ~39kDa
34kDa
26kDa
CAT: ~25kDa
SUMO: ~15kDa
17kDa
Lane 1: Molecular Marker
Lane 2: 1:500
Lane 3: 1:1000
Lane 4: 1:3000
Lane 5: 1:5000
Results
MW
55kDa
43kDa
34kDa
24 hours at 4oC
SUMO-CAT: ~39kDa
26kDa
CAT: ~25kDa
17kDa
SUMO: ~15kDa
Lane 1: Molecular Marker
Lane 2: 1:500
Lane 3: 1:1000
Lane 4: 1:3000
Lane 5: 1:5000
Results
MW
48 hours at 4oC
43kDa
34kDa
SUMO-CAT: ~39kDa
26kDa
CAT: ~25kDa
17kDa
SUMO: ~15kDa
Lane 1: Molecular Marker
Lane 2: 1:500
Lane 3: 1:1000
Lane 4: 1:3000
Lane 5: 1:5000
Conclusions
 Use of sucrose leads to possible protein aggregation
during purification.
 Higher pH levels change protein solubility.
 SUMO-protease is active in cleaving off SUMO from
substrate.
Future Research
 Grow and purify an ICdel (92-261) construct.
 Grow and purify a SUMO ICdel( 92-261)
construct.
 Analyze solubility levels of ICdel modified by
SUMO and solubility levels of ICdel not
modified by SUMO.
Acknowledgements
 Dr. Elisar Barbar
 Dr. Kevin Ahern
 Barbar Lab Staff
 Yujuan Song
 Afua Nyarko
 Justin Hall
 Greg Benison
 Jessica Morgan
 Invitrogen
 HHMI
 URISC
 OSU Biochemistry and Biophysics Dept.