L3: Protein Function and Techniques Flashcards
3 types of Protein Modifications
- Disulfide-bond formation
- Group Addition
- protein phosphorylation
- protein glycosylation
- protein ubiquitination
- Protein modification
How do proteins work to regulate cell functions?
By binding to other molecules (ligands or substrates), small molecules (ions or sugars), or large molecules (proteins, DNA, and RNA)
A protein’s function determines its ____
function:
- structural proteins
- enzymes
- transcription factors
- signal transduction proteins
Proteins with similar function can be grouped together called _____
Protein families
Protein Families
Members of the same protein family that usually have similar structures
Members of protein family have similar _____ and _____
structure and function
Key cell regulators
Enzymes
Suffix “-ase”is usually appended to its name
Enzymes
4 examples of enzymes
- Protease———–degrade protein
- Kinase————–add phosphate group
- Phosphatase—–remove phosphate group
- Ribonuclease—–RNase: degrade RNA
Proteins that catalyze cellular reactions
Enzymes
Substrates Products
tadahhhhh
Enzymes
- converts substrate to product without changing itself
- speed up reactions by factors of 10^6 or more at relatively low temperature
- highly specific and function by lowering the activation energy of the reaction
How is enzymatic activity measured?
Through Vmax and Km
Vmax
- maximum rate of reaction
- the bigger the Vmax, the more active the enzyme
Km
- substrate concentration of 1/2Vmax
- the smaller the Km, the higher the enzyme substrate affinity (better the enzyme)
Antibodies as Molecular Tools
- produced in response to the injection of foreign materials (Antigens)
- bind tightly to specific antigens with > 10^-9 M affinity
- can distinguish among proteins that differ by only a single amino acid
- can be used to determine if a particular protein is present in a complex mixture of proteins
- can be used to purify a particular protein away from the other components of a complex mixture
- can be used to neutralize pathogens
How do we study proteins in living systems?
- Separation
- separating different cellular components and proteins
- centrifugation, chromatography, SDS-PAGE
- Detection (immunological techniques)
- detecting a single protein from a mixture of proteins
- ELISA, Immunoblot, Immunoprecipitation
- Sequence and Structural analysis
- Mass spectometry, X-ray crystallography
Protein purification
to study structure and properties of proteins in interest, it often must be isolated in a pure form
4 steps of Protein separation/purification
- break-up of cells
- physical disruption, Detergent
- Centrifugations
- differential centrifugation
- rate-zonal centrifugation
- Chromatography
- ion exchange
- gel filtration
- affinity column
- Gel Electrophoresis
- SDS-PAGE
- 2D gel
Breaking up cells and tissues
- ULTRASOUND
- break cells with high frequency sound
- DETERGENT
- use mild detergent to make holes in plasma membrane
- FRENCH PRESS
- force cells into small hole using high pressure
- HOMOGENIZE
- when carefully applied, homogenization leave most of the membrane-bounded organelles intact
Differential Centrifugation
- particles that sediment rapidly are separated from slower sedimenting materials that remain in the supernatant (using centrifugal force)
- rapidly sedimenting material is collected in the pellet
- separates a mixture of particles that differ greatly in size
Rate-zonal or velocity centrifugation
- sucrose gradient is used to provide density stability during centrifugation
- particles of similar density sediment based primarily on mass and shape
- provides a finer degree of separation with sucrose density gradient
Column Chromatography
- cylindrical columns filled with water permeable solid of different properties
- different properties behave different in different columns –> allows separation
Column Chromatography (types)
- Ion exchange chromatography
- separate according to charge
- Gel filtration chromatography (or sizing column)
- separate according to size
- Affinity chromatography
- separate according to ability to bind to ligands (affinity)