A Research Style Biochemistry Lab: Collaborating on

1
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
2
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
Cell biology
Organic
Microbiology
Biochemistry
Molecular biology
Inorganic
Physical
Genetics
3
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

4
Research focus

• Hypothesis driven
• Continuity
• Open-ended

Biochemistry Research

• Explorations into the functional or structural
  properties
• of isolated biological molecules under
• controlled conditions

5
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.
6
Cystathionine-b-Lyase (CBL)
Uren, J. R. (1987). "Cystathionine Beta-Lyase
From Escherichia-Coli." Methods In Enzymology
143 483-486.
7
Design implementation

1. Colorimetric assay for product formation
2. Commercially available substrates
3. Complex reaction mechanism
4. Crystal structure

8
Cystathionine-b-Lyase (CBL)
pyridoxal 5-phosphate

• Complex reaction mechanism

• Crystal structure

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.
9
The Process
1) Each student group generates a hypothesis

• analysis of reaction mechanism and enzyme active
  site

I think the hydroxyl group on tyrosine 111
stabilizes substrate binding
10
The Process
2) Each group designs a mutant to test their
hypothesis
11
Mutagenesis with additional silent mutation
CBL DNA
acc aac acc gcc tat gaa ccg agt cag gat
T N T A Y111 E P
S Q D
CBL protein sequence
mutant CBL
T N T A F111 E P
S Q D
mutant DNA
acc aac acc gcc ttt gaa cct agt cag gat
second change introduces or removes a restriction
site, no change in protein sequence silent
mutant
12
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

13
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
Lane 1 1kb DNA ladder Lane 2 5 non-mutant
CBL plasmid DNA Lane 6 mutant CBL plasmid DNA
(Y111F)
Larissa Nordstrom, Chrissie Chow, Rachel Dyer
(2006)
14
The Process
4) Protein expression, isolation and analysis
Affinity chromatography
Bradford assay
SDS-PAGE
15
The Process
5) Enzyme kinetics (functional analysis)

• Three substrates
• Wild-type and mutant enzyme
• Different pH buffers

Experimental Design
measure dP
dt
S ???
Km S at ½ Vmax (Km values from literature)
E ???
S gtgt E
determined through trial and error
16
Results
Group 1
Group 2
CBL
CBL
Km 94 mM kcat 82 sec-1
Km 54 mM kcat 58 sec-1
Y111F CBL
S339A CBL
Km 28 mM kcat 0.81 sec-1
Km 30 mM kcat 0.038 sec-1
17
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
18
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
19
Is this publishable?
20
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
21
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