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Unformatted text preview: MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory
NH2 O P O ONH2 OH
-O N N OO
- N N O O P OO O P OH OH H O H N N kinase
O O O P OH OH H O H N N P O- O H H P O- O H H substrate substrate ATP Why are scientists interested in detecting kinase activity? ADP • Enables monitoring of kinase activity and inhibition • Used for screening of ___________________ • Facilitates the unraveling of complex cell-signalling pathways implicated in
processes ranging from cell cycle regulation to cellular ______________ (including tumor metastasis). Enzyme activity assays detect either product formation or starting material consumption. This is straightforward if a reactant or product has distinct spectroscopic properties (for example, a high extinction coefficient only in the product form.) For kinases this means • Detection of phosphorylated _____________ (product) or • Detection of ______ (product) formation or ______ (starting material) consumption 1 However, phosphorylation is spectroscopically ___________. Neither the formation of phosphorylated product nor the conversion of ATP to ADP can be monitored directly using spectroscopy. The products appear ______________ to the starting materials due to identical absorption features and extinction coefficients. Scientists have overcome this challenge creating a variety of assays. Kinase assays: in buffer, in cell lysate, in vivo Depending on your application, you should consider whether you need an assay that can be performed only with purified enzyme, in a cell lysate mixture, or in vivo. Some other considerations may include a protocol that is: • Easy and safe to use • ____________ versus specific • ________________ (versus an end-point assay). Continuous assays simplify kinetic studies. • A turn-on versus a turn-off assay (some researchers prefer the generation of a positive signal rather than a decrease in signal) • Amenable to high-throughput applications • Sensitive to low levels of kinase activity I. IN VITRO ACTIVITY ASSAYS (FOR PURIFIED KINASES) A) 32P incorporation assay : detection of a radiolabeled, phosphorylated substrate
NH2 O32P OH
-O 32P N N O OO O O P OH OH H O H NH2 N N O O P OO O P OH OH H O H N N N N O H H
-O O- P O- O H H substrate substrate The kinase of interest is incubated with a substrate peptide or protein, radiolabeled [γ-_____]______ and MgCl. Following ______________ of the unreacted [γ-32P]ATP, the incorporation of 32P into the substrate is quantified on a scintillation counter. The requirement for residual ATP separation is the major drawback of this method. Additional assay considerations (both negative and positive): • generates ________________ waste • non-continuous, meaning that it is necessary to take time points if you need kinetic data. • General and sensitive. The assay works for ______ kinase. 2 Note: 32P incorporation (or other in-vitro) assays can be applied to cell lysate if the kinase of interest is first purified from the cell lysate by _________________________. Immunoprecipitation is the use of an _____________ to specifically bind a target protein and provide a handle for protein isolation.
incubate cell lysate w as h substrate + 32P-ATP
32P-substrate + ADP kinase immobilized antibody filter out unreacted 32P-ATP and measure 32P-substrate B) Gel Analysis with a Phospho-specific Antibody: Western blot detection of a phosphorylated substrate Ie. Detection of phosphorylated Crk (an Abl substrate) from cells with increasing concentrations of PDGF (Ting A. Y. et.al. PNAS 98,15003-15008 (2001)). Assay considerations (depending on your specific application) • Non-continuous. • Often used qualitatively • Highly ____________ and often used with crude cell lysate C) Coupled Phosphorylation Assay (Session 13 and 14): (Indirect) detection of ADP formation/ATP consumption Ribo N ADP O HH NH2 ε 340 = 6220 cm-1M-1 Through two enzymatic reactions, Abl kinase activity is coupled to the conversion of __________ to ________. The specific activity of the kinase can be calculated based on the decrease in NADH absorbance at _______ nm over time. The ε340 of NADH = 6220 cm-1M-1. Extinction coefficient (ε): A molecule’s extinction coefficient is a measure of the extent to which the molecule ____________ light at a given wavelength. ε is typically reported for the wavelength of maximum absorbance (εmax) in units of cm-1M-1. 3 Beer’s Law: Abs = εcl where c = concentration, and l = pathlength of the cuvette Beer’s law allows you to use ε to calculate the ___________________ of a sample in solution by measuring the Abs at a given wavelength. Details of the coupled kinase phosphorylation assay The protein kinase catalyzes the transfer of a phosphate group from ATP to a T, S, or Y on the peptide substrate. For our Abl assays, the substrate is EAI___AAPFAKKK.
-O NH3+ OH N O NH HN O NH HN O NH AcHN O O O O H N O N H H N O O N H H N O O NH2 O P OO O NH O N O H N O N H H N O O N H H N O O NH2 +H 3N NH3+ ATP Abl kinase
HN O NH HN O O ________
NH3+ + H3N substrate
O O- NH AcHN O P-substrate O O- Pyruvate kinase (_____) transfers a phosphate group from phosphoenolpyruvate (PEP) to ADP to form _____________ and ATP.
O -O P O OOH O PK OH HO O OH ADP ATP O O PEP pyruvate Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to _____________ with concomitant oxidation of the coenzyme _________ to NAD+.
OH O O
Ribo N ADP O HH NH2 LDH
HO OH O Ribo N+ A DP O H NH2 pyruvate NADH lactate NAD+ You will monitor the ________ of NADH absorption at _____ nm to quantify the __________________ of wt and H396P Abl kinase domain 1. in the absence of any inhibitors 2. with the Abl inhibitor Gleevec 3. with the Abl inhibitor Dasatinib Specific activity in biochemistry is defined as the amount of product formed by an enzyme in a given amount of time. Specific activity is often reported as: _________ (U) per ____ of enzyme, (where 1 unit = 1 µmol of product formed per minute). 4 II. FLUORESCENT PEPTIDE-BASED PROBES Detection of phosphorylated substrate
-O F OH S/T/Y kinase ATP, Mg2+ F O P OO S/T/Y protein or peptide substrate change in the intensity or ! of the fluorescence emission peak F = environment-sensitive or chelation-enhanced fluorophore These probes comprise a substrate peptide that includes an amino acid recognition sequence for a specific kinase and an appropriately positioned _________________sensitive or ______________-enhanced fluorophore. Upon phosphorylation of the substrate probe, the flurophore’s emission properties change, enabling detection. A) Environment-Sensitive Fluorophores The signal intensity and maximum emission wavelength of environment-sensitive fluorophores (also referred to as solvatochromic dyes) are affected by the __________ of the fluorophore’s immediate environment. For example, the fluorophores below are quenched and and red-shifted in polar environments.
NMe2 Dansyl SO3H Acrylodan Me2N NO 2 O R NB D N Me N R N O A phosphorylation-induced conformational change or intramolecular _____________ event that results in a change in the fluorophore’s local environment can be detected by a change in fluorescence intensity.
OH F OPO32- SH2 domain OPO32- kinase ATP, Mg2+ F SH2 domain F substrate (pTyr binding domain) substrate 5 B) Chelation-Enhanced Fluorophores Chelation-Enhanced Fluorophores demonstrate dramatic fluorescence intensity increases upon metal chelation. In CHEF-based kinase activity probes, the probes have low affinity for Mg2+ in the non-phosphorylated form. Once phosphorylated the binding affinity increases drastically, resulting in a ____ to _____ fold fluorescence enhancement.
OH Me2NO2S N O kinase
S OH S/T/Y Me2NO2S N Mg2+ S O OP O O S/T/Y ATP, Mg2+ Protein or peptide substrate with a chelation-sensative fluorophore (ie. “Sox”) This is an ideal assay for cell lysate applications. Adjustments in the sensors to optimize detection with lower levels of MgCl2 (initial conditions required 10 mM MgCl2, above physiological levels) will enable in-vivo applications. Advantages to Peptide-Based Probes • Assays enable continuous readings (no work-up steps prior to detection) making them ideal for kinetic studies. • Peptide probes are amenable to large-scale ______________. • Compared to proteins, peptides are incredibly ________ for handling/storage. • Analogs to sense different kinases and explore different sensing techniques are readily accessible. • Fluorescence change upon phosphorylation in many peptide-based sensors greatly exceeds 100%. Disadvange to Peptide-Based Probes for In-vivo Applications • It is challenging to uniformly introduce peptide probes into cells for in-vivo studies. (Most peptides are not cell-permeable.) Methods to introduce fluorescent probes into cells • ______________________ • Protein transduction domains (typicallyArg-rich peptide sequences) that are fused to target peptides or small proteins and render them cell permeable. • Introduction of a non-natural amino acid during protein expression (hijacking RNA translational machinery) for a genetically-encoded probe. 6 III. IN-VIVO PROTEIN-BASED PROBES (ie. FRET) Protein probes for sensing kinase activity typically employ fluorescence resonance energy transfer (________) to sense a conformational change induced by protein phosphorylation. FRET is a distance-dependent interaction between a donor and an acceptor fluorophore and is used as a “molecular ruler”. When the donor and acceptor fluorophores are in close proximity (10-100 A), the acceptor fluorophores is excited by the donor emission and emits a signal*. *In FRET, the emission spectrum of the donor must overlap with the absorption spectrum of the acceptor. Green Fluorescent Protein (GFP) and related fluorescent proteins • ______-amino acid GFP first isolated from the Jellyfish
Aequorea Victoria • The chromophores of GFP and related fluorescent
proteins arise from natural amino acids, so they can be expressed _____________.
O N H O R2 O cyclization HO H2O OH N R1 NH O N O OH R2 oxidation (slow) HO 2H O N N R1 NH O OH R2 HO R1 HN N H _____65 - _____66 - _____67 For (S65T) GFP: excitation: 488 nm; emission: 507 nm The denatured GFP protein is NOT fluorescent. GFP is _________________-sensitive. Protein engineering has led to the development of a rainbow of other GFP analogs with a range of excitation and emission wavelengths. All are _______________ , but also __________ (~230 amino acids). 7 FRET-based phosphorylation sensors: Detection of a phosphorylation-induced ______________________ change For example, below is a cartoon of a FRET-based sensor for protein tyrosine kinases (PTK) using the CFP/YFP FRET pair. The major advantage to FRET-based probes is that they can be expressed in cells. However, the large ________ of FRET fluorophore pairs is a concern, particularly in comparison to small-molecule fluorophores. Also, most FRET-based probes demonstrate a fluorescence increase of less than _____% following phosphorylation. 8 IV. LABORATORY REPORT GUIDELINES Grading Rubric for the Laboratory Report / Lab Work: 50 points total Laboratory Report: 42 points Abstract: 5 points Introduction: 10 points Explanation of purpose/relevance: (5) Demonstration of understanding: (5) Results: 5 points Organization/Clarity: (5) Discussion: 10 points Interpretation of data: (5) Demonstration of understanding: (5) Appendices: 12 points PyMol Structure Worksheet: (4) Materials and Methods: (4) Supplementary Info: (4) Laboratory Work: 8 points Overall technique: (4) Effort and attitude: (4) Remember: You want to impress us with your understanding of the background, the techniques, and the chemistry involved. ______________________________________________________________________ General Outline of the Module 4 and 5 Laboratory Report: Please use the following subheadings in your report. Abstract This should provide a concise summary of the purpose of the report, major experimental data obtained, and major conclusions. The length should not exceed 250 words. Introduction This serves to introduce the reader to the report that follows. The introduction should also emphasize the relevance and importance of the experiment. Your introduction should include: • Any background information that is required to orient the reader to the experiment. • The purpose of the experiment (why is it important, why is the information helpful/needed) • An overall statement of the scope of the experiment and any of its limitations Do not use the first person. The introduction should include references from any pertinent literature if necessary (the first journal reading assignments from Lectrue #1 should provide sufficient information for your introduction). Please reference as in Nature. Experimental Results 9 Describe the results. Refer the reader to any specific figures, tables, or appendices. All essential figures must go in this section. The goal of this section is to simply report the results. DO NOT discuss or interpret the data here. Discussion (of Results) In this discussion you must interpret the results reported in the section above. State how these results compare with anticipated results or theory. Also, provide suggestions or explanations for any deviations. You may also discuss the sources of any error in this section. Conclusions This is a BRIEF summary of the project goals and major findings. This should be one short paragraph, concise and direct. References Again, please follow Nature reference formatting. It is ok to have only 1 reference. Appendices (MUST have the following three): 1) PyMol structure viewing worksheet (completed in laboratory Session 15) 2) Materials and Methods Include a brief summary of the methods used to obtain the results discussed in the main body of the paper. You should not recopy the entire lab manual protocol. Brevity and clarity are key. For example one might write: Isolation of wt AblK plasmid The wild-type AblK plasmid was isolated from a 12-mL overnight culture of DH5-α cells transformed with the wild-type AblK plasmid. A Qiagen Miniprep kit was used to extract the plasmid from the cells. Final DNA concentration was determined by measuring the absorbance at 260 nm. Protein concentration determination A Bio-Rad BCA assay was used to determine the concentration of the purified, dialyzed AblK protein. The standard curve was obtained using BSA dilutions (0.9, 0.7, 0.55, 0.4, 0.2 ug/uL) and measuring the absorbance at 595 nm. 3) Supplementary Data This section is for any non-essential data/figures that aren’t included in the lab report (such as std curve fits, absorbance readings, sequence information, etc.) There is no length requirement or limit. However, please be as concise and direct as possible while still achieving all of the criteria outlined above. Remember that you want to impress us with your understanding of the background, techniques, and the chemistry involved. 10 ...
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