Biochemistry Exam 2
Covalent Method
(ex: AGT,): Protein of Interest that contains an active-site cysteine, is attached to fluorophore through a reaction catalyzed by AGT Advantages: Large proteins and you can turn on Disadvantages: Cannot turn off
Noncovalent method
(ex: DHFR): Protein of interest is attached to DHFR, which then binds to trimethoprim tightly, catalyzing the addition of the fluorophore to the DHFR Advantages: Large, turn on, and turn off
What are the different methods for incorporating small-molecule fluorophores to proteins and advantages/disadvantages of each?
-Autofluorescent (ex: GFP) -Covalent (ex: AGT,) -Non-Covalent (ex: DHFR) -Peptide Tags: Epitope -Peptide Tags: Tetracysteine and FlAsh, reAsH, and ChoAsH -Peptide Tags: Biotin Ligase System -Quantum Dots
Components required to incorporate an unnatural amino acid using In Vitro tRNA suppression
-Chemically synthesized aminoacyl tRNA with codon for residue of interest -Through Oligonucleotide-directed mutagenesis with a T4 RNA ligase, lines up with plasmid contained tRNA with anticodon to stop codon -In vitro translation results in mutant enzyme with unnatural amino acid site specifically incorporated
Understand protein separation and quantification using 2D gels - DIGE method
-Extract all proteins, fluorescence label, and proceed with 2D gel, quantify by green vs yellow fluorescence -Cut out protein of interest and run through Mass Spectrometry -utilize different fragments in order to identify protein by comparing to database Limitations: 2D Gels are labor intensive -separation is not always great and low resolution -each protein has to be individually extracted from the gel for MS
How is the GFP fluorophore formed and what are ways to change the spectrophotometric properties of GFP?
-GFP is denatured with heat and digested with papain (thiol protease) -resulting chromogenic peptide purified by reverse phase HPLC -the absorption spectrum of the protease fragment is identical to that of the denatured GFP and is pH dependent -Change spectral properties by changing conjugation; you can do this by mutating a natural amino acid, change the residues surrounding the fluorophore, and affect the pi double bond character
How do enzymes affect the activation energy and transition state of a reaction?
-It lowers the activation energy of a reaction and decreases the free energy available at the transition state
Steps involved in evolution of tRNA synthetase and how do positive and negative screens work?
-Make a library of synthetases with random mutations at the DNA level in amino acid bindings pocket -creates a large library of variants, and then use positive and negative screens in order to figure out which mutations allow for the recognition of an unnatural amino acid (X) How it works: 1) Take a tRNA/synthetase from a different organism 2) Change anticodon on tRNA to recognize a stop codon 3) Evolve the tRNA using positive and negative screen 4) create a synthetase mutant that recognizes UAA 5) Add the tRNA synthetase, the mRNA protein encoding protein of interest with stop codon, and add UAA to media 6) Protein is now expressed in cell and is catalytic Positive screen: +UAA -gene for CAT(chloramphenicol acetyl transferase) -figure out the full-length CAT protein that grows in the presence of chloroamphenicol - do not aminoacylate and removes tRNA Negative screen: -UAA -uses barnase and a full length barnase is toxic -Truncated Barnase is a live protein -add natural amino acids -removes tRNA with natural amino acid attached
Components required to incorporate an unnatural amino acid using in vivo tRNA suppression
-Orthogonal tRNA/tRNA synthetase pair -Change to stop anticodon, that should not recognize natural AAs but should still be recognized by tRNA -Selectively changes natural amino acid to unnatural amino acid (X) -evolution of tRNA synthetase
Peptide Tags: Biotin Ligase System
-Protein of Interest undergoes a reaction with biotin ligase and ATP, in which a ketone becomes attaches -the molecule can then be used in a coupling reaction with hydrazine-containing molecules -fluorophore is now attached to protein Advantages: Small, Turn on, Turn off Disadvantages: Needs multiple components; Bir A, ATP, biotin-ketone, and fluorophore-Hydraziole
Peptide Tags: Epitope
-Protein of interest is attached with a peptide epitope tag, and undergoes a reaction with a fluorescently tagged antibody Advantages: Small, turn-on, turn-off Disadvantages: antibody cannot enter the cells and only use for cell surface proteins
Peptide Tags: Tetracysteine and FlAsh, reAsH, and ChoAsH
-Protein of interest is tagged with a tetracysteine and undergoes a reaction with FlAsH, and flurophore is attached Advantages: Turn on and small Disadvantages: No background; only fluorescent when bound to protein -turn off slow
How to use FRET to establish assays to monitor enzyme activity, and protein-protein interactions?
-Utilize FRET when two fluorophores are located within 100 angstroms of each other -the emission spectrum of one fluorophore (donor) must overlap the excitation spectrum of the second fluorophore (acceptor) -FRET can be used for confirming direct binding partners; attach one FP to the "Bait" protein and the FRET partner to each other possible "prey" proteins
Quantum Dots
-can be targeted to cell surface proteins using biotin ligase Advantages: Great spectral properties Disadvantages: Large, needs primary, secondary Ab to connect to POI
fluorescence
-excitation by photon of light and emission of light (short lifetime)
What are the different classes of enzyme inhibitors and how they modulate Km and Vmax, and what is signified by a Ki value?
1) Competitive Reversible Inhibitors; competes with substrate for binding to enzyme active site, no change with Vmax and Km increases as more substrate is required to reach half of Vmax 2) Non-competitive reversible inhibitor; binds to a site separate from substrate, Vmax decreases and no change with Km 3)Irreversible Inhibitors; Ex: Lactic Antibiotics
What are the mechanisms by which an enzyme accelerates a reaction?
1) Proximity and Orientation Effects 2) Preferential Binding at the Transition state; Transition state stabilization 3) Acid-Base Catalysis 4) Covalent Catalysis 5)Metal-ion Catalysis
Lineweaver Burke equation
1/v = (Km/Vmax)(1/[S]) + 1/Vmax
How to use fluorescence to develop metal ions and post translational modifications?
Calmodulin: a genetically-encoded protein-based calcium sensor -different chemical sensors for Post-Translational Methods, including Phosphorylation, Methylation, Acetylation, Ubiquitylation, and Hydroxylation Phosphorylation: First, phosphorylate a peptide sequence using kinase and complete a FRET assay. There is an increase in FRET when there is phosphorylation Histone Methylation: Utilizes a protein that will only bind to the modified sequence and observe a percent emission ratio change in the methylation site mutant
Native Chemical Ligation
Combine chemical and biosynthesis of proteins to generate large proteins with unnatural amino acids
if given a single purified full-length protein, what are the steps involved in determining the primary sequence?
Edmond Degradation -first protein separation through 2d-gel electrophoresis -then, peptide separation through liquid chromatography
FRET (flu
Energy transfers from donor molecules, without emission, to acceptor molecules when they form dipole dipole interactions. 3 main requirements: 1. they have to be in the right orientation 2. they have to be really close (efficiency of the FRET is proportional to the reciprocal of the distance btw. the donor and the acceptor by the sixth power!) 3. spectral overlap btw. donor and acceptor. application: protein-protein interactions investigation
Understand when and how to use ICAT; What are the limitations of the method?
ICAT -> Isotope-coded affinity tag -First, Extract proteins and label cysteines with isotopic label -cysteine-reactive groups with isotopically-labels light or heavy -peptide separation will occur and quantify through MS1 and identify through MS2 Limitations: Only quantifies cysteine-containing peptides, and if protein has no cysteines, no quantification can occur
chemi-lumin
Light emitted as a result of a chemical reaction not related to a biological substance Typically involves redox reactions and can happen at room temperature
first excited state
S1, and the absorption of photon from the ground state to this first excited state
Understand when and how to use SILAC; What are the limitations of the method?
SILAC -> Stable isotope labeling of amino acids in cell culture -metabolically incorporate Lys/Arg-heavy vs light -extract proteins and then mix -digest in trypsin and proceed with peptide separation through c18 chromatography -Utilize mass spectrometry once to quantify light vs heavy isotopes -complete mass spec a second time in order to identify fragments Limitations: Cannot be used for primary human samples and is very expensive
How does a peptide fragment in the mass spectrometer: what are the different fragment ions that are formed?
The most common peptide fragments observed in low energy collisions are a, b and y ions, as described in the figure above. The b ions appear to extend from the amino terminus, sometimes called the N-terminus, and y ions appear to extend from the carboxyl terminus, or C-terminus.
Michaelis Menten equation
V=Vmax[S]/Km+[S] Vmax=maximal rate of the enzyme-catalyzed reaction when [ES]= [ET]-total enzyme Km is the substrate concentration at which rate 1/2(Vmax)
quenching flu
a decrease in fluorescence intensity due to one of several processes such as excited state reactions, energy transfer, complex formation, and collisional quenching. In sensing, the main mode of quenching occurs when a quencher molecule interacts with a fluorophore.
fluorophore
a fluorescent chemical compound
triplet state
an electronic state in which there are two unpaired electrons
Glycosidic linkage
bond connecting anomeric carbon to another sugar
phosphoresce
continuous emission of light from a substance requiring neither heat nor sustained exposure to radiation (long lifetime)
quantum yield
efficiency of fluorophore, and equals the number of photons out/number of photons in
Understand when and how to use iTRAQ; What are the limitations of the method?
iTRAQ-isobaric tag for relative and absolute quantification -extract proteins and digest in trypsin -label amine-groups (N-term + Lys) with isotopic label -mass balance the isotopic label (reporter group) with the amine-reactive group -MS1 contains only one peak and cannot quantify -MS2 will fragment and quantify -iTRAQ is able to multiplex many samples, up to 16 Limitations: High cost
Luminescenc
light emitted by means other than burning, such as chemical or biochemical action or radiation
Stokes shift theory for fluorescence
the fluorescence emission occurs at a longer wavelength than the incident light
ground state
the lowest allowable energy state of an atom
bioluminescence
the production of light by means of a chemical reaction in an organism
Lifetime
time from wavelength excitation to wavelength emission
non-radiative decay
transfer excitation energy to the vibrational levels without any photon emission