Lecture 9 Studying Protein Structure and Function Flashcards
What is the purpose of Protein Characterization?
the process of determining the structure and function of a protein
how do you characterize a protein?
studying the protein’s interactions with its environment, ligands, and other proteins.
What are the different types of spectroscopy used in protein characterization? (6) ICXNCF
Infrared (IR) Spectroscopy
Circular Dichroism (CD) Spectroscopy
X-ray Crystallography
Nuclear Magnetic Resonance (NMR)
Spectroscopy
Cryo-Electron Microscopy
Fluorescence Spectroscopy
how does Infrared Spectroscopy work?
Uses infrared radiation to study bond vibrations.
what is the frequency of CH bonds like in Infrared Spectroscopy?
high frequency
what is the frequency of peptide bonds like in Infrared Spectroscopy?
can vary based on hydrogen bonding.
what level of protein structure is Infrared Spectroscopy good for studying?
secondary structures
how does Circular Dichroism (CD) Spectroscopy work?
Uses polarized light to analyze protein secondary structures.
what is the absorbance peak of Beta sheets in Circular Dichroism?
217 nm
what is the absorbance peak of Alpha helices in Circular Dichroism?
195 nm
what are the steps of the X-ray Crystallography process?
Purify the protein
Form a crystal.
Expose to x-rays.
Analyze diffraction patterns to infer structure.
how does Nuclear Magnetic Resonance (NMR) Spectroscopy work?
Studies protein structure by analyzing nuclear spin in a magnetic field.
what setting for proteins is Nuclear Magnetic Resonance most effective?
Effective for studying proteins in solution
what protein characterization method is not good for studying proteins in a solution?
crystallography
how does monitoring enzyme activity help to study proteins?
to understand protein function
what method is used to predict 3D protein structure?
AlphaFold
how does AlphaFold work?
use amino acid sequences to predict a protein’s 3D structure.
How does Cryo EM work?
Proteins are flash-frozen on a copper grid and subjected to a beam of electrons
The electron diffraction pattern reveals the protein structure
what does Cryo-Electron Microscopy do?
determine the structure of proteins in solution, especially large proteins and complexes.
how does 3D Image Reconstruction work?
Multiple images of the protein are taken from different angles, and a 3D structure is reconstructed.
what is a Cryo-EM Example involving Proteasome, what is the job of proteasome?
Cryo EM helps determine the structure of large protein complexes like proteasomes, which break down proteins for recycling.
how is AlphaFold used in terms of Ligands?
AlphaFold can predict interactions between proteins and ligands, useful for designing protein-ligand studies.
what are the pros of Cryo-EM?
No crystallization
realistic image
what is a pro of X-ray crystallography?
High resolution
what is a con of X-ray crystallography?
needs crystals
what are the pros of NMR spectroscopy?
Good for smaller proteins
shows interactions
what methods were used to study SARS CoV-2 Spike Protein during COVID-19 and why?
Cryo EM and X-ray crystallography were used to determine the structure of the spike protein and its interaction with ACE2.
how does Fluorescence Spectroscopy work?
A technique to study protein structure using chromophores, which absorb and emit light at different wavelengths.
what is the purpose of Green Fluorescent Protein?
to visualize proteins inside cells
how does Green Fluorescent Protein work?
absorbs and emits light.
what is the Green Fluorescent Protein?
a jellyfish protein
how does Tryptophan Fluorescence work?
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.
