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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

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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


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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


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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


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Research focus:

  • Hypothesis driven

  • Continuity

  • Open-ended

Biochemistry Research

  • Explorations into the functional or structural properties

  • of isolated biological molecules under

  • controlled conditions


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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.


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Cystathionine-b-Lyase (CBL)

Uren, J. R. (1987). "Cystathionine Beta-Lyase From Escherichia-Coli." Methods In Enzymology143: 483-486.


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Design implementation:

  • Colorimetric assay for product formation

  • Commercially available substrates

  • Complex reaction mechanism

  • Crystal structure


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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 Biology262(2): 202-224.


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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”


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The Process:

2) Each group designs a mutant to test their hypothesis


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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


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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


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1

2

3

4

5

6

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)


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The Process:

4) Protein expression, isolation and analysis

Affinity chromatography

Bradford assay

SDS-PAGE


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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


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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


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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


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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



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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


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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


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