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
Joseph Chihade
Department of Chemistry
Carleton College
The origins of this project…
Curricular Goals:
• Expose students to fundamental techniques
• Reinforce concepts from lecture
• Teach experimental design and data
interpretation
A “Research-Style” lab:
• Continuity throughout the term
• Hypothesis driven
• Open-ended
Some institutional differences:
Carleton
St. Olaf
Many of the students in the
Many students have considerable
course are chemistry majors with experience with techniques in
little, if any, molecular biology lab molecular biology.
experience.
Course is taught once a year to
students who are co-enrolled in
Biological Chemistry course.
Course is taught twice a year.
Several students are alumni of
Biochemistry course.
8-9 lab periods per term.
13-14 lab periods per term
What we needed:
A gene for an enzyme:
A structure:
The opportunity:
Genome sequencing
From: National Center for Biotechnology Information
Availability of structural data
From: San Diego Supercomputer Center
Modular design:
1.
Structural Biology: Students use protein structure and enzymatic
reaction mechanism to form hypotheses, design mutations.
2.
Molecular biology: Students use site directed mutagenesis to create
mutants and analyze restriction patterns to check success.
3.
Biochemistry: Students purify mutant protein and assess effect of
mutation by comparing kinetics with mutant and wildtype enzymes.
Or…
3.
Last year’s work
Biochemistry: Students purify mutant protein and assess effect of
mutation by comparing kinetics with mutant and wildtype enzymes.
1.
Structural Biology: Students use protein structure and enzymatic
reaction mechanism to form hypotheses, design mutations.
2.
Molecular biology: Students use site directed mutagenesis to create
mutants and analysis of restriction patterns to check success.
Next year’s work
Our choice: Cystathionine-b-Lyase (CBL)
NH3+
-O
O
S
O
O-
+ H2O
NH3+
Cystathionine
CBL
O
O
-O
+ NH4+ + HS
O
Pyruvate
Clausen, T., R. Huber, et al. (1996). "Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase
from Escherichia coli at 1.83 angstrom." Journal of Molecular Biology 262(2): 202-224.
ONH3+
Homocysteine
Biological/Medical Relevance:
Activated methyl cycle and methionine biosynthesis
Defects in the methionine pathway
Elevated homocysteine ➔ increased ROS ➔ arteriosclerosis
Part 1: Make a hypothesis.
On what basis do students formulate a hypothesis?
• Analysis of the enzyme’s structure.
• Knowledge about the chemical mechanism.
• A database of results from previous students’ work
at both colleges.
The value of a naïve hypothesis.
• We strive to go with students’ choices.
• Sometimes “daring” mutations are the most
interesting ones.
An example:
K210
S339
H2N
OH
H2N
HN
COO
R372
H2N
H
COO
H3N
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”
K210
S339
H2N
OH
H2N
HN
COO
R372
H2N
H
COO
H3N
OH
S
W340
N
Y111
H
G86
OH
O
H
PO3
Y56
N
HN
CH3
H
HO
OH
OH
H3C
OH
Y338
Y238
T209
Part 2: Make a mutant.
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
•Compare restriction digests of wild type and mutant DNA
*silent mutation adds a restriction site
Bfa I digest of plasmid DNA
WT Potential mutants
Larissa Nordstrom, Chrissie Chow, Rachel Dyer (2006)
Part 3: Characterize the mutant.
Affinity chromatography to purify protein
QuickTime™ and a
TIFF (Uncomp resse d) de com press or
are nee ded to s ee this picture.
Assessment of purity
Quantitation
Absorbance
Bradford Protein Assay
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
Enzyme kinetics:
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
Putting it together.
•Revisit hypothesis
“I think the hydroxyl group on tyrosine 111
stabilizes substrate binding”
•Evaluate calculations
70 fold reduction 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 we’ve learned
Units are still a stumbling block.
Never underestimate the difficulty of a
simple calculation
Initial “quick and dirty” assay can help students
gain perspective.
Tension between standardization and open ended
writing in reporting results.
There is a bridge across the river.
The value of collaboration
Technical help is just a phone call away!
Pedagogical conversation is too!
Building up a database of knowledge more
quickly than we otherwise would.
Students can build on each other’s hypotheses.
There is a bridge across the river.
So, can you publish this data? We think so!
• Need replication and validation of results
Students who are interested continue work as
independent project
Both models allow replicates to be run in
subsequent years.
Is this really like research?
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 pretty much exactly what we did in Chem 321 lab.”
- Paul Letendre ‘05, on his work as a research
technician at Mayo after graduation.
Overall sense of empowerment and ownership of their mutants
Acknowledgements
Carleton College, Department of Chemistry
Spring 04-05
Alice Agyiri
Andrew Berry
Stephan Chapman
Stephanie Contag
Mark Ericson
Katie Freeman
Sara Ganaha
Vickie Gunderson
David Jackson
Spring 05-06
Emily Johnson
Erin Addison
Paul Letendre
Disan Davis
Robbie McDermott Thayne Dickey
Thaibinh Nguyen
Jon Dozier
Claire Richardson Michael Draper
Kartik Sampath
Randy Foss
Sinele Tsabedze
Dani Hargreaves
Kate Waller
Admire Kuchena
Dorissa Zemirah
Won Lee
Alex Marston
Cam Noland
Shayna Simmons
Matt Topeff
Pete Watson
Ellen Valkevich
Amanda Winters
Will VanStory
Lauren Wagner
Chris Ward
Maya Warren
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