This codebook.txt file was generated on 2022-01-03 by wilsonkm ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset Binding interaction between PKA-C and RKIP and its mutant P74L 2. Author Information Principal Investigator Contact Information Name: Gianlugi Veglia Institution: University of Minnesota Email: vegli001@umn.edu Associate or Co-investigator Contact Information Name: Cristina Olivieri Institution: University of Minnesota Email: colivier@umn.edu 3. Date of data collection: 2015-07-01 to 2020-12-30 4. Geographic location of data collection (where was data collected?): BMBB Department, Structural Biology Division, University of Minnestota Twin-Cities, Minneapolis, Minnesota, MN 55455 5. Information about funding sources that supported the collection of the data: NIH grant GM100310 to Gianluigi Veglia -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: CC0 1.0 Universal 2. Links to publications that cite or use the data: Raf Kinase Inhibitory Protein (RKIP) Regulates the cAMP-dependent Protein Kinase Signaling Pathway Through a Positive Feedback loop (under revision - PNAS) 3. Recommended citation for the data: Olivieri, Cristina; Veglia, Gianluigi. (2022). Binding interaction between PKA-C and RKIP and its mutant P74L. Retrieved from the Data Repository for the University of Minnesota, https://doi.org/10.13020/39bz-0r13. --------------------- DATA & FILE OVERVIEW --------------------- 1. File List A. Filename: 200717_PKA_RKIP.dataset Short description: Raw data form BLItz of the kinetics of binding of RKIP to immobilized Human-His-tagged PKA-C (apo form) B. Filename: 200721_RKIP_PKA-ATPgN.dataset Short description: Raw data form BLItz of the kinetics of binding of RKIP to immobilized Human-His-tagged PKA-C in presence of ATP analog (PKA-C/ATPgN saturated) C. Filename: Blitz_201109.pzfx Short description: BLItz Processed data the kinetcks of binding of RKIP to apo and ATPgN-saturated PKA-C D. Filename: Chemical_shift_list_PKA_RKIP_P74L.xlsx Short description: Chemical shift perturbation(CSP) of the amide residues for RKIPwt and P74L upon the addition of PKA-C and CSP for PKA-C amide in complex with RKIPwt E. Filename: Chemical_shift_perturbation.pzfx Short description: Chemical shift perturbation(CSP) of the amide residues for RKIPwt and P74L upon the addition of PKA-C and CSP for PKA-C amide in complex with RKIPwt and list file Note on .pzfx files: .PZFX files can be viewed using GraphPad Prism viewer. 2. Relationship between files: A and B are the raw data acquired using the BLItz instrument (in the format that the machine use) while C contains the same data sets but exported in an excel file. D contains the chemical shift lists and the tab for calculating the chemical schift perturbations, while E contains only the latest and it has been used to generate the CSP figures for the pubblication 3. Additional related data collected that was not included in the current data package: 1H-15N HSQC Spectra of RKIP/PKA-C complex, P74L/PKA-C complex 4. Are there multiple versions of the dataset? no If yes, list versions: Name of file that was updated: i. Why was the file updated? ii. When was the file updated? Name of file that was updated: i. Why was the file updated? ii. When was the file updated? -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. Description of methods used for collection/generation of data: From Material and Methods of the pupplication (link to be given) BLI experiments were performed using a BLItz biosensor system (ForteBio, Pall, Singapore) at 27 °C. Uncleaved His(6x)-PKA-C (1 μM) was resuspended in 20 mM MOPS (pH 6.5), 90 mM KCl, 10 mM MgCl2, 10 mM DTT, and 1 mM NaN3, and immobilized on the Ni-NTA (NTA) Biosensors (ForteBio, Pall, Singapore). RKIPWT was dialyzed in the same buffer, diluted to a final concentration of 280 μM, and applied in a dose-dependent manner (1:35,1:70,1:140, 1:280 molar ratio) to the biosensor containing the immobilized PKA-C. For the binding of RKIPWT to nucleotide-saturated/PKA-C, the buffer was supplied with 2 mM of ATPγN (2000 times excess), to prevent the phosphorylation of RKIPWT. Triton-X (0.05%) was also added to the buffer to avoid non-specific protein interaction. Parallel experiments were performed for reference where the sensor was titrated with buffer to the immobilized PKA-C (with or without ATPγN) and these resulting signals were subtracted during data analysis. The association and dissociation periods were set to 300 and 450 seconds, respectively. The data were analyzed using the software provided by the instrument and plotted using GraphPad Prism nine software package (GraphPad Software, Inc). All the experiments were performed in triplicate to determine the experimental error. NMR experiments. NMR measurements were performed on a Bruker Avance spectrometer operating at a 1H Larmor frequency of 700 MHz equipped with a cryogenic probe or on a Bruker Avance III 850 MHz spectrometer equipped a TCI cryoprobe. All data were acquired at 300 K. The typical NMR sample contained 20 mM KH2PO4 (pH 6.5), 90 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM NaN3, and 10% D2O. The concentrations of U-15N labeled RKIPWT and P74L mutant were 100 μM. For the NMR titration, a standard [1H-15N] HSQC pulse sequence was used, and five titration points (1:0, 1:0.1, 0:0.4,1:1,1:1.5, and 1:2 RKIP:PKA-C/ATPγN molar ratio) were collected. The total number of scans were 48, with 2048 and 128 complex points for 1H and 15N, respectively. All data were processed using NMRPipe(39) and visualized using NMRFAM-Sparky. Chemical shift perturbations (CSPs) were measured using amide proton and nitrogen resonances for each residue as previusly reported. Standard [1H, 15N]-TROSY-HSQC experimentwas used to record the amide fingerprint spectra of PKA-C/ATPγN/RKIPWT ternary complex. The total number of scans were 96, with 2048 and 128 complex points for 1H and 15N, respectively. The sample consisted of 100 μM 15N-PKA-C, 0.12 mM unlabeled-RKIPWT (1:1.2 molar ratio) and 120 time excess of ATPγN (12 mM). 2. Methods for processing the data: The BLItz data were analyzed using the software provided by the manufacturing company (ForteBio, Pall, Singapore). The NMR spectra was analysed using NMRPipe and Sparky. 3. Instrument- or software-specific information needed to interpret the data: BLItz™ Label-Free Protein Analysis System, ForteBio (Singapore). The software for analyzing the data is given by the company upon request. Topship (Bruker) used to acquire the NMR spectra, given by the company upon request. NMRPipe can be download here:https://spin.niddk.nih.gov/NMRPipe/install/legacy.html NMRFAM-SParky can be downloaded here: https://nmrfam.wisc.edu/nmrfam-sparky-distribution/ 4. Standards and calibration information, if appropriate: Read the methods section 5. Environmental/experimental conditions: Read the methods section 6. Describe any quality-assurance procedures performed on the data: Read the methods section 7. People involved with sample collection, processing, analysis and/or submission: Dr. Cristina Olivieri, Dr. Jonggul Kim