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How to Study Protein Structure?

How to Study Protein Structure?

• Protein Purification

• Structure Elucidation

• Primary Structure

• Secondary and Tertiary Structure

• Quaternary Structure

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Protein purification

Tissue / cell lysate

preparation

Protein purification
Protein purification by column chromatography

Size-exclusion

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Protein purification
Protein purification by affinity chromatography

Alberts, Essential Cell Biology, 3rd ed.

Protein purification
Confirmation of Protein Purity

Lodish H., Berk A., Matsudaira P., Kaiser CA, Krieger M, Scott MP,
Zipursky L, Darnell J. Molecular Cell Biology, 5th ed.

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Structure Elucidation
Primary Structure Elucidation by protein digestion and mass spectrometry (Fingerprinting)

Graves et al., Microbiol. Mol. Biol. Rev. March 2002 vol. 66 no. 1 39-63

MS exploits principle of separating ions according to mass to charge ratio

- mass and charge determine the time it takes to reach the detector
- large peptides slower; highly charged faster

Structure Elucidation
Primary Structure Elucidation by protein digestion and mass spectrometry (Fingerprinting)

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Structure Elucidation
Primary Structure Elucidation by protein digestion and mass spectrometry (Fingerprinting)

Predicted peptides (example)

Structure Elucidation

Identification of known protein

Graves et al., Microbiol. Mol. Biol. Rev. March 2002 vol. 66 no. 1 39-63

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Structure Elucidation
Primary Structure Elucidation by protein digestion and mass spectrometry (Fingerprinting)

Liquid chromatography–mass spectrometry (LC-MS / HPLC-MS)

How to detect unknown protein?

http://www.physiologie.uni-freiburg.de/research-techniques/mass-spectrometry

Tandem mass spectro attached to the output of an HPLC dsystem

Structure Elucidation
LC-MS/MS

Allows to detect
precursor Sequencing
product
ions
unknown peptides

https://www.slideshare.net/Nabiilah/mass-spectrometry-in-pharmacognosy

Frederick Klink

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Structure
Elucidation

Protein Sequence

Structure Elucidation

ER beta_N

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Structure Elucidation
Secondary and Tertiary Structures elucidation by X-ray crystallography and NMR
X-ray crystallography

Nuclear Magnetic Resonance (NMR)

small volume of concentrated protein solution atomic nuclei, particularly hydrogen nuclei, have a magnetic
that is placed in a strong magnetic field; moment
or spin: that is, they have an intrinsic magnetization, like a bar
magnet 8
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Structure Elucidation

Different protein Backbone Ribbon

models

Wire Space-filling

Alberts, Essential Cell Biology, 3rd ed.

Backbone - basic amino acid sequence stx; clean way for comparing
Ribbon - visualize alpha beta domains
Wire - highlights side chains, useful for determining function (enzyme)
Space Filling - feel of the protein shape

Structure Elucidation

Different protein models

Lodish H., Berk A., Matsudaira P., Kaiser CA, Krieger M, Scott MP,
Zipursky L, Darnell J. Molecular Cell Biology, 5th ed.

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Structure Elucidation

How to study quaternary structures?

In non-reducing buffer (no BME) Baotic et al., 2013, American Journal of Physiology - Heart and Circulatory Physiology 305 (2) H219-H227

What if Protein Expression is Too Low?

Clone the sequence

Vector that express protein
Increase amount of protein
Purify the proteins

Why E coli used as host?

- Prokaryotic
simultaneous transcription and translastion
- Glycolysation of Golgi

https://www.genwaybio.com/technologies/protein-expression

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TAKE HOME ASSIGNMENT – Group activity

On a piece of paper, write the name of your groupmates who participated

A thesis student wants to study a protein homolog of Cyclin D1 in a newly discovered

species. Cyclin D1 contains a cyclin box domain for cyclin-dependent kinase binding
that is conserved across species.

Draw a schematic diagram on how the student will be able to determine the protein
structure of Cyclin D1 in the newly discovered species. Describe your diagram as a
figure legend.

Students will also be graded individually during recitation next meeting

Read about

PROTEIN REGULATION and FUNCTION

NO Class on Sept 19

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