a research style biochemistry lab: collaborating on the integration of research and teaching at two...
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A Research Style Biochemistry Lab: Collaborating on the Integration of Research and Teaching at Two Institutions
Gregory W. MuthDepartment of Chemistry
St. Olaf College
Joe ChihadeDepartment of Chemistry
Carleton College
HistoricalBiochemistry:
1828 synthesis of urea1833 isolation of amylase1896 fermentation using yeast extracts1903 general acceptance of the term “biochemistry”1962 ACS publication of Biochemistry1998 ASC biochemistry requirement
Interdisciplinary aspects:
Biochemistry
Organic
Inorganic
Physical Genetics
Molecular biology
Analytical Cell biology
Microbiology
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:
•Explorations into the functional or structural properties of isolated biological molecules under controlled conditions
•Hypothesis driven•Continuity•Open-ended
Biochemistry Research
Design implementation:
Cystathionine--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.
Defects in methionine pathway•elevated homocysteine•increased ROS•arteriosclerosis
Activated methyl cycle and methionine biosynthesis
Cystathionine--Lyase (CBL)
Uren, J. R. (1987). "Cystathionine Beta-Lyase From Escherichia-Coli." Methods In Enzymology 143: 483-486.
OS
O
NH3
NH3
O
OH2O
CBLO H3C
O
NH3 NH4O
O
O
pyruvatehomocysteinecystathionine
+ +SH
Design implementation:
1. Colorimetric assay for product formation2. Commercially available substrates3. Complex reaction mechanism4. Crystal structure
Cystathionine--Lyase (CBL)
N+ CH3
OH
OH
H
OP
O
O
O-
-
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.
The Process
1) Each student group generates a hypothesis
H3N SCOO
H
N
H
O
PO3
OH
CH3
NH
COO
K210S339
R372
W340
T209
G86
H2NOH
HN
H2N
H2N
HN
H3C
HO
H
OH
Y56
OH
Y338
OH
Y238
OH
Y111
“I think the hydroxyl group on tyrosine 111 stabilizes substrate binding”
•analysis of reaction mechanism and enzyme active site
The Process:
2) Each group designs a mutant to test their hypothesis
H3N SCOO
H
N
H
O
PO3
OH
CH3
NH
COO
K210S339
R372
W340
T209
G86
H2NOH
HN
H2N
H2N
HN
H3C
HO
H
OH
Y56
OH
Y338
OH
Y238
OH
Y111
CBL DNA
CBL proteinsequence
mutant CBL
acc aac acc gcc tat gaa ccg agt cag gat
T N T A Y111 E P S Q D
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
Mutagenesis with additional silent mutation
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
•Compare restriction digests of wild type and mutant DNA*silent mutation adds a restriction site
•Standard kit isolation
1 2 3 4 5 6
Lane 1: 1kb DNA ladderLane 2 – 5: non-mutant CBL plasmid DNALane 6: mutant CBL plasmid DNA
Bfa I digest of plasmid DNA
Larissa Nordstrom, Chrissie Chow, Rachel Dyer (2006)
(Y111F)
The Process:4) Protein expression, isolation and analysis
Bradford assay SDS-PAGE
Affinity chromatography
Bradford Protein Assay y = 0.0283x + 0.0064
R2 = 0.9992
00.10.20.30.40.50.60.70.8
0 5 10 15 20 25 30
Concentration (mcg/ml)
Ab
sorb
ance
The Process:
5) Enzyme kinetics (functional analysis)
•Three substrates•Wild-type and mutant enzyme•Different pH buffers
Experimental Design:
[E] = ???
measure d[P]dt
Km = [S] at ½ Vmax (Km values from literature)
[S] = ???
determined through trial and error
[S] >> [E]
Results:
Km = 54 Mkcat = 58 sec-1
Km = 28 Mkcat = 0.81 sec-1
CBL
Y111F CBLCBL-Y111F
0
0.3
0.60.9
1.2
1.5
1.8
0 0.02 0.04 0.06 0.08 0.1
[cystathionine] (mM)
Vo
(mcM
/min
)
CBL
0
5
10
15
20
25
30
0 0.02 0.04 0.06 0.08 0.1[cystathionine] (mM)
Vo
(mcM
/min
) Group 1 Group 2
Km = 94 Mkcat = 82 sec-1
CBL
Km = 30 Mkcat = 0.038 sec-1
S339A CBL
The Process:
6) Each group shares results in a final presentation or report
•Revisit hypothesis
•Evaluate calculations
70 fold change in kcat , minimal change in Km
“I think the hydroxyl group on tyrosine 111 stabilizes substrate binding”
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
•Conclude
Lessons learned
Units, units, units!!!!
Perspective – how much is reasonable? when is a change significant?
Always provide a standard template for reporting results
Never underestimate the difficulty of a simple calculation
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
St. Olaf College, Faculty and Students
Fall 05-06Brennan DeckerKiyomi GotoMike KuprianColin ReilyHayley RossChris Torstenson
Spring 05-06Nisar BaigChrissie ChowRachel DyerChristine GilleLiz JohnsonMatt MajerusBrandon MoriartyLarissa Nordstrom
Fall 06-07Andrew BodgerColette CaveTyler DrakeSultan MirzoyevJames MorrisonPat NelsonPaul NicholKatherine OysterRyan Ritzer
Carleton College, Department of Chemistry