chapter 3 - semantic scholar...purifying, detecting, and characterizing proteins aa protein must be...
TRANSCRIPT
![Page 1: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/1.jpg)
Chapter 3
Protein Structure and Function
![Page 2: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/2.jpg)
Broad functional classes
So Proteins have structure and function... Fine!
-Why do we care to know more????
Understanding functional architechture gives us POWERto:•Diagnose and find reasons for diseases•Create modifying drugs•Engineer our own designer-proteins
![Page 3: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/3.jpg)
DNA
(mRNA)
Translation:
Translation into 3D structure:
3D structure determines function:
Modifications:Chemical modification of aminoacids
Interaction with other molecules
Proteolytic cleavage
(Location)
New 3D structure
New function
Proteins are single, unbranched chains of amino acid monomersThere are 20 different amino acidsThe amino acid sidechains in a peptide can become modified, extending the functional repetoire of aminoacids to more than hundred different amino acids.A protein’s amino acid sequence determines its three-dimensional structure (conformation) In turn, a protein’s structure determines the function of that proteinConformation (=function) is dynamically regulated in several different ways
Protein structure determines function
![Page 4: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/4.jpg)
All amino acids have the same general structure but the side chain (R group) of each is different
Cα
R: Hydrophilic:
Basic
Acidic
Non-charged
Hydrophobic
“Special”
![Page 5: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/5.jpg)
Hydrophilic amino acids
![Page 6: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/6.jpg)
Hydrophobic and “special” amino acids
![Page 7: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/7.jpg)
Backbone
Side-chains
Peptide bonds connect amino acids into linear chains
![Page 8: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/8.jpg)
Side chain modifications change the chemical (functional) properties of proteins
Glycosylation
Ubiquitylation
=> Expanding the repetoire of existing amino acid side-chains to > 100 variations!
Acetylation
Phosphorylation
Hydroxylation
Methylation
Carboxylation
![Page 9: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/9.jpg)
Four levels of structure determine the shape of proteins
Primary: the linear sequence of amino acids peptide bonds
Secondary: the localized organization of parts of a polypeptide chain (e.g., the α helix or β sheet)backbone hydrogen bonds
Tertiary: the overall, three-dimensional arrangement of the polypeptide chainhydrophobic interactions, hydrogen bonds (non-covalent bonds in general) and sulfur-bridges
Quaternary: the association of two or more polypeptides into a multi-subunit complex
![Page 10: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/10.jpg)
Primary and secondary structure (example: hemagglutinin)
β-strand α-helix
![Page 11: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/11.jpg)
Secondary structure
α Helixβ Sheet
β (U)-turn
![Page 12: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/12.jpg)
Motifs are regular combinations of secondary structures. Motifs form domains!
Three examples of Motifs from different types of DNA-binding proteins
Tertiary structure
![Page 13: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/13.jpg)
Structural, functional or topological domains are modules of secondary and tertiary structure
Globular domain
Tertiary structure
Each of these proteins contain the EGF globular domain.
- But each of these proteins have a different function
Tertiary structure
![Page 14: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/14.jpg)
Different graphical representations of the same protein(tertiary structure)
![Page 15: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/15.jpg)
Quaternary structure
![Page 16: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/16.jpg)
Multiprotein complexes: molecular machines
![Page 17: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/17.jpg)
Sequence homology suggests functional and evolutionary relationships between proteins
When the stucture of a newly discovered protein is known, comparison to other proteins across species can help predict function
![Page 18: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/18.jpg)
Folding, modification, and degradation of proteins
The life of a protein can briefly be described as: synthesis, folding, modification, function, degradation.
A newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final protein
All molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the molecule
Most proteins have a limited lifespan before they are degraded (turn-over time)
![Page 19: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/19.jpg)
Aberrantly folded proteins are implicated in slowly developing diseases
An amyloid plaque in Alzheimer’s disease is a tangle of protein filaments
![Page 20: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/20.jpg)
The information for protein folding is encoded in the sequence
![Page 21: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/21.jpg)
Folding of proteins in vivo is promoted by chaperones
Large proteins with a lot of secondary structure may require assisted folding to avoid aggregation of unfolded protein
- Molecular chaperones and chaperonins prevent aggregation of unfolded protein
![Page 22: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/22.jpg)
Folding of proteins in vivo is promoted by chaperones
Large proteins with a lot of secondary structure may require assisted folding to avoid aggregation of unfolded protein
- Chaperones and chaperonins prevent aggregation of unfolded protein
![Page 23: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/23.jpg)
Functional design of proteins
Protein function often involves conformational changes
Proteins are designed to bind a range of molecules (ligands)Binding is characterized by two properties: affinity and specificity
Antibodies and enzymes exhibit precise ligand/substrate-binding specificityBut can have variable affinities
Enzymes are highly efficient and specific catalystsAn enzyme’s active site binds substrates(ligands) and carries out catalysis
![Page 24: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/24.jpg)
Antibody/antigen interaction: an example for ligand-binding with high affinity and specificity
![Page 25: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/25.jpg)
Enzymes have high substrate affinity sites and catalytic sites
![Page 26: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/26.jpg)
Kinetics of an enzymatic reaction are described by Vmax and Km
![Page 27: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/27.jpg)
Kinetics of an enzymatic reaction are described by Vmaxand Km
![Page 28: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/28.jpg)
Enzymes in one pathway can be physically associated
![Page 29: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/29.jpg)
Mechanisms that regulate protein activity
Altering protein synthesis rate and proteasomal degradation
Allosteric transitionsRelease of catalytic subunits, active inactive states, cooperative binding of ligands
Chemical modification: Phosphorylation, acetylation etc. dephosphorylation, deacetylation etc.
Proteolytic activation
Compartmentalization
![Page 30: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/30.jpg)
Protein degradation via the ubiquitin-mediated pathway
Cells contain several other pathways for protein degradation in addition to this pathway
ATP
![Page 31: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/31.jpg)
Allosteric transitions: Cooperative binding of ligands
Sigmoidal curve indicates cooperative binding (of ligands, substrates, ca ions) in contrast to standard Michaelis-Menten Kinetics
![Page 32: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/32.jpg)
Conformational changes induced by Ca2+ binding to calmodulin
Cooperative binding of calcium: binding of one calcium enhances the affinity for the next calcium
When 4 calcium are bound a major allostericconformational changeoccurs
Calmodulin is a switch protein because this effect in turn regulates other proteins bound by the compact calmodulin
![Page 33: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/33.jpg)
Another class of switch proteins: GTPases
![Page 34: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/34.jpg)
Chemical modification
Example: Phosphorylation dephosphorylation
![Page 35: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/35.jpg)
Proteolytic cleavage of proinsulin to produce active insulin
![Page 36: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/36.jpg)
CompartmentalizationExample:Membrane proteins
Each cell membrane has a set of specificmembrane proteins that allows themembrane to carry out its activitiesMembrane proteins are either integralor peripheralIntegral transmembrane proteins containone or more transmembrane α helicesPeripheral proteins are associated withmembranes through interactions withintegral proteins
![Page 37: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/37.jpg)
Schematic of membrane proteins in a lipid bilayer
![Page 38: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/38.jpg)
Mechanisms that regulate protein activity
Altering protein synthesis rate and proteasomal degradation
Allosteric transitionsRelease of catalytic subunits, active inactive states, cooperative binding of ligands
Chemical modification: Phosphorylation, acetylation etc. dephosphorylation, deacetylation etc.
Proteolytic activation
Compartmentalization
![Page 39: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/39.jpg)
Example containing all levels of regulatin of protein activity
GFP-tagged GLUT4
![Page 40: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/40.jpg)
Now that you KNOW the basic principles of protein structure and function you can UNDERSTAND:
Protein and ProteomeAnalytical techniques
![Page 41: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/41.jpg)
Purifying, detecting, and characterizing proteins
A protein must be purified to determine its structure and mechanism of action
Detecting known proteins can be usefull for diagnostic purposes
Molecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties (size, mass, density, polarity, affinity...)
Elementary toolbox includes: centrifugation, electrophoresis, liquid chromatography (LC), spectrometry, ionization/radiation. -applied in various advanced forms and combinations.
![Page 42: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/42.jpg)
Centrifugation can separate molecules that differ in mass or density
![Page 43: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/43.jpg)
Electrophoresis separates molecules according to their charge:mass ratio
SDS-polyacrylamidegel electrophoresis
Even coating of proteins allows even charge distribution -> larger mass = higher total charge
![Page 44: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/44.jpg)
Two-dimensional electrophoresis separates molecules according to their charge and their mass
![Page 45: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/45.jpg)
Highly specific enzymes and antibody assays can detect individual proteins
Immunoblot (= Western Blot) based on affinity
![Page 46: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/46.jpg)
Liquid chromotography (LC):
Separation of proteins by size: gel filtration chromatographyAdd mobile phase: buffer
Stationary phase:
![Page 47: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/47.jpg)
Separation of proteins by charge: ion exchange chromatography
Also: Reversed-phase LC: separation by hydrophobicityStationary phase: non-polar, Mobile phase: moderately polar
![Page 48: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/48.jpg)
Separation of proteins by specific binding to another molecule: affinity chromatography
![Page 49: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/49.jpg)
Proteomics, the analysis of complex protein mixtures
Genome databases allow prediction of genes -> protein primary structureEach protein can be fragmented into peptides which are composed of aa’s.Each aa has a unique mass to charge ratio at a given pHEach protein therefore has a unique peptide-fingerprint
Technique: proteins->peptides->mass/charge ratio measurement -> compare against whole proteome (genome based) database -> identify proteins
![Page 50: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/50.jpg)
Time-of-flight mass spectrometry measures the mass of proteins and peptides
Matrix-Assisted-Laser-Desorption/Ionization Time-of-flight mass spectrometry (MALDI-TOF MS)
![Page 51: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/51.jpg)
MS spectrum
![Page 52: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/52.jpg)
Example of a proteome analysis workflow
Cell/tissue of interest
Isolate organelles (fractionation)
Confirm organelle-specific proteins
Subfractionate, detect peptides, identify corresponding proteins
![Page 53: Chapter 3 - Semantic Scholar...Purifying, detecting, and characterizing proteins aA protein must be purified to determine its structure and mechanism of action aDetecting known proteins](https://reader036.vdocuments.site/reader036/viewer/2022071216/60471b06a9f4e64ab06f0505/html5/thumbnails/53.jpg)
X-ray crystallography is used to determine protein structure
Other techniques such as cryoelectron microscopy and NMR spectroscopy may be used to solve the structures of certain types of proteins