A Research Style Biochemistry Lab: Collaborating on
the Integration of Research and Teaching at Two Institutions
Gregory W. Muth
Department of Chemistry
St. Olaf College
Joe Chihade
Department of Chemistry
Carleton College
HistoricalBiochemistry:
1828 synthesis of urea
1833 isolation of amylase
1896 fermentation using yeast extracts
1903 general acceptance of the term “biochemistry”
1962 ACS publication of Biochemistry
1998 ASC biochemistry requirement
Interdisciplinary aspects:
Analytical
Organic
Cell biology
Microbiology
Biochemistry
Molecular biology
Inorganic
Physical
Genetics
Curricular goals:
•Explore fundamental biochemistry techniques
•Teach experimental design and data interpretation
•Expose chemistry students to interdisciplinary pedagogy
•Make connections between molecular structure and function
•Reinforce concepts from lecture
Research focus:
•Hypothesis driven
•Continuity
•Open-ended
Biochemistry Research
•Explorations into the functional or structural properties
of isolated biological molecules under
controlled conditions
Design implementation:
Activated methyl cycle and methionine biosynthesis
Defects in methionine pathway
•elevated homocysteine
•increased ROS
•arteriosclerosis
Cystathionine-b-Lyase (CBL)
(E. coli)
Steegborn, C., et al., Kinetics and inhibition of recombinant
human cystathionine gamma-lyase – Toward the rational
control of transsulfuration. Journal of Biological Chemistry,
1999. 274(18): p. 12675-12684.
Cystathionine-b-Lyase (CBL)
O
NH3
S
O
O
NH3
cystathionine
O
+
H2O
O
O
CBL
SH
O
NH3
homocysteine
O
+
H3C
O
NH4
pyruvate
Uren, J. R. (1987). "Cystathionine Beta-Lyase From Escherichia-Coli." Methods In Enzymology 143: 483-486.
Design implementation:
1.
2.
3.
4.
Colorimetric assay for product formation
Commercially available substrates
Complex reaction mechanism
Crystal structure
Cystathionine-b-Lyase (CBL)
O
H
O
-O P O
O-
OH
+
N
H
CH3
pyridoxal 5’-phosphate
Complex reaction mechanism
Crystal structure
Clausen, T., R. Huber, et al. (1996). "Crystal structure of the pyridoxal5'-phosphate dependent cystathionine beta-lyase from Escherichia coli
at 1.83 angstrom." Journal of Molecular Biology 262(2): 202-224.
The Process
1) Each student group generates a hypothesis
•analysis of reaction mechanism and enzyme active site
K210
S339
H 2N
OH
H2 N
HN
COO
R372
H2 N
H
COO
H 3N
OH
S
W340
N
Y111
H
G86
OH
O
H
PO3
Y56
N
HN
CH3
H
HO
OH
OH
T209
H3C
OH
Y338
Y238
“I think the hydroxyl group on tyrosine 111
stabilizes substrate binding”
The Process:
2) Each group designs a mutant to test their hypothesis
K210
S339
H 2N
OH
H2 N
HN
COO
R372
H2 N
H
COO
H 3N
OH
S
W340
N
Y111
H
G86
OH
O
H
PO3
Y56
N
HN
CH3
H
HO
OH
OH
H3C
OH
Y338
Y238
T209
Mutagenesis with additional silent mutation
CBL DNA
acc aac acc gcc tat gaa ccg agt cag gat
CBL protein
sequence
T
N
mutant CBL
T
mutant DNA
acc aac acc gcc ttt gaa cct agt cag gat
N
T
T
A
Y111 E
P
S
Q
D
A
F111 E
P
S
Q
D
second change introduces or removes a restriction site,
no change in protein sequence – silent mutant
Advantages of silent mutation
•Use of bioinformatics software (EMBOSS)
•Review genetic code (protein
DNA)
•Predict outcome of restriction digests (NEB cutter 2.0)
•Avoid the “black box” of DNA sequencing
•Students empowered to “order” DNA oligomer
and restriction enzyme
The Process:
3) DNA isolation and analysis
•Standard kit isolation
•Compare restriction digests of wild type and mutant DNA
*silent mutation adds a restriction site
Bfa I digest of plasmid DNA
1 2
3
4
5 6
Lane 1:
Lane 2 – 5:
Lane 6:
1kb DNA ladder
non-mutant CBL plasmid DNA
mutant CBL plasmid DNA
(Y111F)
Larissa Nordstrom, Chrissie Chow, Rachel Dyer (2006)
The Process:
4) Protein expression, isolation and analysis
Affinity chromatography
Bradford assay
Absorbance
Bradford Protein Assay
SDS-PAGE
y = 0.0283x + 0.0064
R2 = 0.9992
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
Concentration (mcg/ml)
25
30
The Process:
5) Enzyme kinetics (functional analysis)
•Three substrates
•Wild-type and mutant enzyme
•Different pH buffers
Experimental Design:
measure d[P]
dt
[S] = ???
Km = [S] at ½ Vmax (Km values from literature)
[E] = ???
[S] >> [E]
determined through trial and error
Results:
CBL
Vo (mcM/min)
30
25
20
15
Group 2
CBL
Km = 54 mM
kcat = 58 sec-1
Km = 94 mM
kcat = 82 sec-1
10
5
0
0
0.02
0.04
0.06
0.08
0.1
[cystathionine] (mM)
Vo (mcM/min)
Group 1
CBL
CBL-Y111F
1.8
1.5
1.2
0.9
0.6
0.3
0
Y111F CBL
S339A CBL
Km = 28 mM
Km = 30 mM
kcat = 0.81 sec-1 kcat = 0.038 sec-1
0
0.02
0.04
0.06
0.08
[cystathionine] (mM)
0.1
The Process:
6) Each group shares results in a final presentation or report
•Revisit hypothesis
“I think the hydroxyl group on tyrosine 111
stabilizes substrate binding”
•Evaluate calculations
70 fold change in kcat , minimal change in Km
•Conclude
The placement of Y111 within the active site (distant
from PLP) along with the kinetic data suggest that the
Y111 hydroxyl helps position the substrate in an
optimal orientation for the chemical reaction
Lessons learned
Units, units, units!!!!
Never underestimate the difficulty of a simple calculation
Perspective – how much is reasonable?
when is a change significant?
Always provide a standard template for reporting results
There is a bridge across the river
Is this publishable?
Student Perceptions:
“Experimental Biochem. Lab does apply to the real world!!!!”
- Hayley Ross ’07, while doing summer research
at the University of Pittsburgh
“I do exactly what we did in Chem 321 lab”
-from a student who worked as a research
tech at Mayo after graduation.
Overall sense of empowerment and ownership of their mutants
Acknowledgements
Carleton College, Department of Chemistry
St. Olaf College, Faculty and Students
Fall 05-06
Brennan Decker
Kiyomi Goto
Mike Kuprian
Colin Reily
Hayley Ross
Chris Torstenson
Spring 05-06
Nisar Baig
Chrissie Chow
Rachel Dyer
Christine Gille
Liz Johnson
Matt Majerus
Brandon Moriarty
Larissa Nordstrom
Fall 06-07
Andrew Bodger
Colette Cave
Tyler Drake
Sultan Mirzoyev
James Morrison
Pat Nelson
Paul Nichol
Katherine Oyster
Ryan Ritzer