Lecture 9 Studying Protein Structure and Function

Question 1

What is the purpose of Protein Characterization?

Answer 1

the process of determining the structure and function of a protein

Question 2

how do you characterize a protein?

Answer 2

studying the protein’s interactions with its environment, ligands, and other proteins.

Question 3

What are the different types of spectroscopy used in protein characterization? (6) ICXNCF

Answer 3

Infrared (IR) Spectroscopy

Circular Dichroism (CD) Spectroscopy

X-ray Crystallography

Nuclear Magnetic Resonance (NMR)
Spectroscopy

Cryo-Electron Microscopy

Fluorescence Spectroscopy

Question 4

how does Infrared Spectroscopy work?

Answer 4

Uses infrared radiation to study bond vibrations.

Question 5

what is the frequency of CH bonds like in Infrared Spectroscopy?

Answer 5

high frequency

Question 6

what is the frequency of peptide bonds like in Infrared Spectroscopy?

Answer 6

can vary based on hydrogen bonding.

Question 7

what level of protein structure is Infrared Spectroscopy good for studying?

Answer 7

secondary structures

Question 7

how does Circular Dichroism (CD) Spectroscopy work?

Answer 7

Uses polarized light to analyze protein secondary structures.

Question 8

what is the absorbance peak of Beta sheets in Circular Dichroism?

Answer 8

217 nm

Question 9

what is the absorbance peak of Alpha helices in Circular Dichroism?

Answer 9

195 nm

Question 10

what are the steps of the X-ray Crystallography process?

Answer 10

Purify the protein

Form a crystal.

Expose to x-rays.

Analyze diffraction patterns to infer structure.

Question 11

how does Nuclear Magnetic Resonance (NMR) Spectroscopy work?

Answer 11

Studies protein structure by analyzing nuclear spin in a magnetic field.

Question 12

what setting for proteins is Nuclear Magnetic Resonance most effective?

Answer 12

Effective for studying proteins in solution

Question 13

what protein characterization method is not good for studying proteins in a solution?

Answer 13

crystallography

Question 14

how does monitoring enzyme activity help to study proteins?

Answer 14

to understand protein function

Question 14

what method is used to predict 3D protein structure?

Answer 14

AlphaFold

Question 15

how does AlphaFold work?

Answer 15

use amino acid sequences to predict a protein’s 3D structure.

Question 16

How does Cryo EM work?

Answer 16

Proteins are flash-frozen on a copper grid and subjected to a beam of electrons

The electron diffraction pattern reveals the protein structure

Question 16

what does Cryo-Electron Microscopy do?

Answer 16

determine the structure of proteins in solution, especially large proteins and complexes.

Question 17

how does 3D Image Reconstruction work?

Answer 17

Multiple images of the protein are taken from different angles, and a 3D structure is reconstructed.

Question 18

what is a Cryo-EM Example involving Proteasome, what is the job of proteasome?

Answer 18

Cryo EM helps determine the structure of large protein complexes like proteasomes, which break down proteins for recycling.

Question 19

how is AlphaFold used in terms of Ligands?

Answer 19

AlphaFold can predict interactions between proteins and ligands, useful for designing protein-ligand studies.

Question 20

what are the pros of Cryo-EM?

Answer 20

No crystallization

realistic image

Question 21

what is a pro of X-ray crystallography?

Answer 21

High resolution

Question 22

what is a con of X-ray crystallography?

Answer 22

needs crystals

Question 23

what are the pros of NMR spectroscopy?

Answer 23

Good for smaller proteins shows interactions

Question 24

what methods were used to study SARS CoV-2 Spike Protein during COVID-19 and why?

Answer 24

Cryo EM and X-ray crystallography were used to determine the structure of the spike protein and its interaction with ACE2.

Question 25

how does Fluorescence Spectroscopy work?

Answer 25

A technique to study protein structure using chromophores, which absorb and emit light at different wavelengths.

Question 26

what is the purpose of Green Fluorescent Protein?

Answer 26

to visualize proteins inside cells

Question 27

how does Green Fluorescent Protein work?

Answer 27

absorbs and emits light.

Question 28

what is the Green Fluorescent Protein?

Answer 28

a jellyfish protein

Question 29

how does Tryptophan Fluorescence work?

Answer 29

amino acid tryptophan absorbs UV light and emits fluorescence Its wavelength changes depending on whether it is buried inside or exposed on the protein surface.