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)
Ion exchange chromatography
- separate molecules according to charge
- (+) – come through column first –> (-) bind the beads and be removed later
- (-) – opposite
Gel Filtration Chromatography
- separates molecules according to size
- larger molecules migrate faster in gel filtration column
- smaller molecules migrate slower
- take longer to travel through pores of beads
SDS-PAGE
- sodium dodecyl sulfate polycrylamide gel electrophoresis
- separated according to Molecular Weight on polyacrylamide gels
- only separate proteins with large differences in Molecular Weight
Immunoblot (Western blot)
- can detect specific protein within a crude extract based on the ability of an antibody to selectively bind the protein of interest
Immunoblot (Western blot) - 2
- separate protein in an SDS-PAGE gel
- Blot protein bands to a nitrocellulose membrane
- incubate membrane with Ab1 that recognizes the antigen. then Ab2 or protein A that recognizes Ab1
- visualize antigen by either radioactivity label of Ab2 or protein A, or a colorimetric reactions catalyzed by the marker enzyme conjugated to Ab2 or protein A
Chromatin Immunoprecipitation
can detect protein association with DNA in cell
Proteins can also be detected using _____ to specific proteins and secondary antibodies that have a _____ _____
antibodies, flourescent tag
Amino acids in membrane are _____
hydrophilic
Amino acids in cytoplasmic domain are _____ …….
positively charged and interact with negatively charged and interact with negatively charged head groups
Isolation of membrane proteins require ……….
screening of detergents to find the optimal conditions to isolate proteins in tact from membrane
Isoelectric focusing (IEF) gel electrophoresis
- based on electric charge, which is determined by the numbers of acidic and basic residues but not the molecular weight of a protein
- charge proteins will migrate through the pH gradient until they reach their pI (isoelectric point)–pH at which the net change of the protein is 0
- 2 unrelated proteins having similar masses are likely to have identical charges
- low pH = (+) protein
- high pH = (-) protein
- isoelectric point = protein has no net charge
- -> no longer migrates in electric field
- isoelectric point = protein has no net charge
2D gel electrophoresis
- 1st dimension: Isoelectric focusing (IE) –> separation by charge
- proteins migrate in a pH gradient until they rich a point where pH = isoelectric point (0 net charge) of protein
- 2nd dimension: SDS-PAGE –> separation by size
- -proteins migrate according to size
3 Protein sequence analysis
- chemical method – Edman degradation
- mass spectrometry
- deduction from DNA sequence
Edman degradation
- only good fro small peptides and non-modified peptides
1. react with phenylisothiocyanate on free amino group
2. acid cleavage
3. amino acid analysis
4. repeat many times
Mass spectrometry (MS)
- fast and accurate way to measure molecular weight (MW)
- predicts ratio of mass to charge which determines MW
Mass spectrometry (4 steps)
- laser desorption/ionisation
- atom ionized by knocking one or more electrons off to give a (+) ion
- MS always works with (+) ions
- acceleration
- ions are accelerated so they all have the same kinetic energy
- deflection
- ions are deflected by magnetic field according to their mass
- lighter = more they are deflected
- amount of deflection depends on number of (+) charges on ion (how many e- were knocked off in the 1st stage)
- more ion is charged = more deflected
- detection
- beam of ions passing through machine is detected electrically
Proteomics
- study of all proteins produces in cell or species under a given set of conditions
- ex: 2D gel
- electrophoresis coupled with MS to identify MW of protein
X-ray Chrystallography
- X-ray diffraction patters of protein crystals allow determination of protein structure to atomic resolution
- yields an electron density map that illustrates the position and types of atoms
Why study protein structure?
- analysis of 3D structure of HLH type transcription factor helps in understanding hot protein dimer binds to DNA
