SC thanks the Whitehead Summer Research Fellowship. MJS thanks the Startup Funds and the Faculty Development Fund at the University of San Francisco. MCH acknowledges funding from the National Institutes of Health (NIH MIRA 5R35GM133684-02) and the National Science Foundation (NSF CAREER 2048265).

1. Electronic absorption spectroscopy: direct titration with buffer competition
<ol>
	<li>Sample preparation
	<ol>
		<li>Prepare a buffered solution of 50 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (bisTris) at pH 7.4 using ultrapure water (&#62;18 M&#937; resistance). Remove trace metal ions by incubating with a high-affinity resin for at least 2 h with subsequent filtration.</li>
		<li>Dissolve or dilute a known quantity of the peptide into the metal-free buffer. 
		NOTE: When monitoring d-d bands with small extinction coefficients21, higher concentrations of peptide must be used. Here, the final concentration of C-peptide in buffered solution was 300 &#181;M (volume depends on the size of the cuvette). C-peptide was synthesized by solid-phase peptide synthesis and is detailed elsewhere in literature14.</li>
		<li>Dissolve a known mass of CuCl2 in ultrapure water to make a solution at 10-15 mM. 
		NOTE: It is important to initially dissolve the metal salt in nonbuffered water to prevent precipitation. Other Cu(II) salts may be used, but care must be taken to ensure that the anion is weakly coordinating.</li>
	</ol>
	</li>
	<li>Running the experiment
	<ol>
		<li>Turn on the electronic absorption spectrophotometer and let it warm up for ~15-20 min before use. Launch the spectrophotometer software and configure the parameters such as scanning range (200-900 nm), scan rate (200 nm/s), and double beam baseline-corrected (more parameters are listed in the Supplemental File).</li>
		<li>Collect a baseline with no cuvettes or samples in the beam paths.</li>
		<li>Using two matched cuvettes in the double-beam spectrophotometer, load one cuvette with 115 &#181;L of ultrapure water and the other cuvette with 115 &#181;L of the peptide sample. Ensure that there are no air bubbles in the cuvettes as these will interfere with the signal.</li>
		<li>Place the cuvette with ultrapure water in the reference beam and the cuvette with peptide in the sample beam.</li>
		<li>Collect the absorption spectrum of the metal-free (apo) peptide.</li>
		<li>Add in a sub-stoichiometric amount (0.5 equivalents, 150 &#181;M) of the Cu(II) solution into the cuvette with the peptide sample. Ensure that the volume of Cu(II) added is less than 3 &#181;L and record the volume for analysis later.</li>
		<li>Gently pipette up and down to mix the solution while avoiding the generation of air bubbles. Let the solution react and equilibrate for 5 min and record the absorption spectrum.</li>
		<li>Repeat the addition of Cu(II) aliquots as in step 1.2.6 into the peptide solution for the following equivalents: 1.0, 1.5, 2.0, 3.0, and 5.0 (or a total of 300, 450, 600, 900, and 1,500 &#181;M). Make sure to record the total volume of Cu(II) added and the total volume of the cuvette. 
		NOTE: If more resolution is desired, reduce the spacing between equivalents.</li>
		<li>Remove the sample cuvette and thoroughly clean as per the manufacturer&#39;s directions.</li>
		<li>Add the buffered solution without peptide and record the absorption spectrum. Repeat the addition of Cu(II) aliquots as in steps 1.2.5-1.2.8, recording the absorption spectrum for each Cu(II) equivalent.</li>
		<li>Export all spectra as csv files for processing. Thoroughly clean the cuvettes according to the manufacturer&#39;s instructions and power down the spectrophotometer.</li>
	</ol>
	</li>
	<li>Processing the data
	<ol>
		<li>Load all spectra on a spreadsheet program.</li>
		<li>Subtract the buffer-only (0 &#181;M Cu(II)) spectrum from every other spectrum to remove any absorbance features from the buffer itself.</li>
		<li>Normalize each spectrum to account for the dilution resulting from the addition of the Cu(II) solution (step 1.2.6). See the Supplemental File, Eq (1)14 for an example of the normalization where vinitial is the volume (115 &#181;L) of peptide added to the cuvette, vCu(II) is the volume of Cu(II) solution added in step 1.2.6, and Absbuffer subtracted spectrum is the data obtained in step 1.2.7.</li>
		<li>Graph all spectra together to identify regions of change. 
		&#8203;NOTE: Typical d-d bands from Cu(II) complexes range from 500 to 750 nm. This spectrophotometric titration can be challenging due to the small extinction coefficient from d-d bands, which are Laporte-forbidden transitions in octahedral geometry21. If the absorbance is too weak, an alternative approach is to utilize chromophoric ligands that result in charge transfer bands upon binding to Cu(II) (see section 2).</li>
	</ol>
	</li>
</ol>
2. Electronic absorption spectroscopy: peptide competition with chromophoric ligand
<ol>
	<li>Sample preparation
	<ol>
		<li>Dissolve 1,10-phenanthroline (phen) in ultrapure water to obtain a final concentration of ~1 mM.</li>
		<li>In addition to the sample preparation described in section 1.1 for the peptide (step 1.1.2) and Cu(II) (step 1.1.3), prepare a 10 &#181;M Cu(II) and 40 &#181;M phen solution in buffer ([Cu(phen)3]2+). Ensure that the volume fills the cuvette.</li>
	</ol>
	</li>
	<li>Running the experiment
	<ol>
		<li>Start the electronic absorption spectrophotometer as in steps 1.2.1 and 1.2.2 but set the scanning range to 200-400 nm.</li>
		<li>In two matched cuvettes, load one cuvette with 115 &#181;L of ultrapure water and the other cuvette with 115 &#181;L of the [Cu(phen)3]2+ solution. Place the cuvette with water in the reference beam and the cuvette with [Cu(phen)3]2+ solution in the sample beam.</li>
		<li>Collect the absorption spectrum of the metal-ligand complex.</li>
		<li>Add a stoichiometric amount (&#8776;1 equivalents, &#8776;10 &#181;M) of peptide to the [Cu(phen)3]2+ solution. Gently pipette up and down to thoroughly mix but be careful not to introduce air bubbles. Record the volume of peptide added for future analysis. 
		NOTE: Using cuvettes that hold 115 &#181;L, adding 3.83 &#181;L of 300 &#181;M peptide yields a final peptide concentration of 9.7 &#181;M.</li>
		<li>Incubate the solution for 5 min to reach equilibrium. Record the absorption spectrum. 
		NOTE: If the binding affinity of the metal-peptide and metal-ligand are similar, the concentration of peptide added will need to be in large excess. Be sure to account for the total volume of peptide added for normalization.</li>
		<li>Repeat the addition of peptide aliquots into the [Cu(phen)3]2+ solution for the following approximate equivalents: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 22, and 26. Record the volume of peptide added so that the diluted concentration can be determined.</li>
		<li>Remove the sample and clean the cuvette thoroughly according to the manufacturer&#39;s instructions. Collect a spectrum of the buffer. Collect a spectrum of 50 &#181;M peptide in the buffer.</li>
		<li>Export all data as csv files for processing and clean the cuvettes according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Processing the data
	<ol>
		<li>Load the spectra on a spreadsheet program and subtract the buffer spectrum from the other spectra.</li>
		<li>Normalize the spectra following Supplemental File, Eq (2)14.</li>
		<li>Using the extinction coefficient for [Cu(phen)3]2+ at 265 nm (&#949;max = 90,000 M-1cm-1)22, determine the concentration of [Cu(phen)3]2+ with each addition of the peptide.</li>
		<li>For each addition of the peptide, determine the concentration of the Cu(II)-peptide complex by subtracting the remaining concentration of [Cu(phen)3]2+, as determined in step 2.3.2, from the initial concentration of [Cu(phen)3]2+ (at 0 &#181;M peptide).</li>
		<li>Calculate the concentration of free phen ligand by the equation in Supplemental File, Eq (3)14.</li>
		<li>Calculate the concentration of free peptide using Supplemental File, Eq (4)14, where [peptide]stock represents the undiluted peptide titrated into the cuvette, V1 represents the volume of stock peptide added, V2 is the total volume of the cuvette, and [Cu2+-peptide] is determined in step 2.3.4.</li>
		<li>Calculate the experimental binding affinity (Kex) using Eq (5) in Supplemental File23.</li>
		<li>Relate the dissociation constant of Cu(II)-peptide to Kex by Eq (6)23 in Supplemental File, where Kd,Cu(II)-phen = 1.0 &#215; 10-9 (see 22). Determine the average and standard deviation from all the determined dissociation constants. 
		&#8203;NOTE: Under a 4:1 ratio of phen:Cu(II), [Cu(phen)3]2+, [Cu(phen)2]2+, and [Cu(phen)]2+ exist in the solution, and the peptide will chelate Cu(II) away from the species ([Cu(phen)]2+) with the weakest binding22.</li>
	</ol>
	</li>
</ol>
3. Isothermal titration calorimetry
<ol>
	<li>Sample preparation
	<ol>
		<li>Prepare a buffered solution of 15 mM 3-morpholinopropane-1-sulfonic acid (MOPS) at pH 7.4 using ultrapure water (&#62;18 M&#937; resistance). Remove trace metal ions by incubating with a high-affinity resin for at least 2 h with subsequent vacuum filtration through a bottle-top 0.45 &#181;m membrane.</li>
		<li>Dissolve a known mass of CuCl2 in ultrapure water to prepare a solution of &#8776;50-100 mM. Dilute this Cu(II) solution into buffer to obtain a 1.0 mL solution with final concentration of 1.4 mM Cu(II). Record the exact volume of the CuCl2 solution used.</li>
		<li>Dissolve or dilute the peptide solution in buffer to make 450 &#181;L of a 154 &#181;M peptide solution. Ensure that the same proportion of additional ultrapure water from step 3.1.2 is added to the peptide solution, which will reduce heat of dilution and increase signal-to-noise.</li>
		<li>After preparation of the samples, ensure that the solutions are at the same pH and, if needed, adjust accordingly.</li>
		<li>Optional step: Degas the solutions to minimize microbubbles loaded into the ITC.</li>
	</ol>
	</li>
	<li>Running the experiment
	<ol>
		<li>Turn on the ITC. Launch the ITC software to run the instrument. Wait for the first initialization, which will ask to rehome the buret; then, follow the instructions on the screen.</li>
		<li>Remove the cover from the reference cell. Remove any water from the reference cell and rinse three times with 450 &#181;L of degassed ultrapure water.</li>
		<li>Slowly draw up ultrapure water to the 450 &#181;L mark of a loading syringe, taking care not to introduce air bubbles into the syringe. Insert the loading syringe into the reference cell until it is &#8776;1 mm from the bottom, and slowly inject part of the solution until 150 &#181;L remains in the loading syringe. Move the loading syringe plunger quickly up and down by &#8776;25 &#181;L several times to dislodge any bubbles on the cell surface. Slowly inject until the plunger reaches the 100 &#181;L mark on the loading syringe, thereby dispensing a total of 350 &#181;L of ultrapure water into the reference cell, and replace the reference cell cover.</li>
		<li>Remove any residual solution from the sample cell and load with 450 &#181;L of 10 mM ethylenediaminetetraacetic acid (EDTA) using a loading syringe. Soak for 10 min to ensure that trace metal ions are removed because the EDTA will bind trace metals.</li>
		<li>Remove the EDTA solution (with bound trace metal ions) and thoroughly rinse the loading syringe with copious amounts of ultrapure water.</li>
		<li>Clean the ITC in accordance with the manufacturer&#39;s directions, rinsing the sample cell with ultrapure water.</li>
		<li>Condition the sample cell by rinsing with 450 &#181;L of buffer at least three times.</li>
		<li>Remove the buffer that is conditioning the sample cell. Load the peptide solution into the sample cell using the loading syringe (follow step 3.2.3).</li>
		<li>Rinse the titration syringe with 200 &#181;L of buffered solution. To do this, remove the plunger and use a micropipette to pipette the buffer through the hole at the top of the glass titration syringe, through the syringe, and out the needle below.</li>
		<li>Fully insert the plunger into the titration syringe.</li>
		<li>Dip the tip of the titration syringe needle into the metal solution and slowly pull the plunger up, causing the metal solution to fill the syringe and result&#160;in a void volume at the top of the glass part of the titration syringe. Remove most of the void volume by rotating the titration syringe parallel to the floor, remove the plunger, and slightly tilt the glass part toward the floor. Give the titration syringe a gentle shake so that the solution moves to the end of the glass part of the titration syringe and fills most of the void volume, but ensure that 2-3 &#181;L of void volume remains. While keeping the syringe parallel to the floor, reinsert the plunger.</li>
		<li>Hold the titration syringe upright, dip the tip of the needle back into the metal solution, and push the plunger down until air ceases to come out of the needle. Load the titration syringe by slowly pulling up the plunger to just above the 50 &#181;L mark while keeping the tip of the needle in the solution.</li>
		<li>Carefully insert the glass part of the titration syringe into the buret and screw until finger-tight. When a small amount of solution comes out of the titration syringe due to compression of the plunger, use a light-duty delicate wiper to carefully absorb the solution without touching the tip of the needle.</li>
		<li>Insert the buret with titration syringe into the sample cell and fasten it securely.</li>
		<li>Set up the parameters on the ITC software. Starting on Instrument Control, set the stirring rate (typical stirring rates range from 150 to 350 RPM) and the temperature at which the experiment will be conducted (typically 25 &#176;C). Enter the syringe and cell concentrations in millimolar units under Experiment Details.</li>
		<li>In the Experiment Method section, select Incremental Titration. Click on Setup and specify 20 injections of 2.5 &#181;L. If more resolution is required to observe the binding event, increase the number of injections and decrease the volume per injection. Input the time spacing between each injection so that it is long enough for the signal to equilibrate and return to baseline, typically 300 s.</li>
		<li>Click the run button to start the experiment and specify where the data are saved.</li>
		<li>Upon completion of the experiment, clean the sample cell and titration syringe.</li>
		<li>Run all experiments in at least triplicate to ensure precise data collection.</li>
		<li>Run a control experiment where the metal solution is titrated into the buffered solution (in the absence of peptide) to ensure the heat of dilution from the metal ion is small and that there are no unaccounted-for equilibria. If the heat of dilution is large, consider a different buffer system, if possible.</li>
	</ol>
	</li>
	<li>Processing the data
	<ol>
		<li>Launch the ITC analysis software and load the data file for analysis.</li>
		<li>Navigate to the Baseline tab and inspect the thermogram. Note any exogenous heat evolved or absorbed from air bubbles or other artifacts in the thermogram. Look for spikes that are not due to injection of the metal solution.</li>
		<li>Ensure that the baseline generated by the analysis software follows the portion of the data after injection and equilibration. If it deviates, use Baseline Pivot Points to adjust the baseline. Ensure that the Integration Regions include the peak generated from injection of the metal, but occlude any air bubbles or artifacts in the thermogram found in step 2. Subtract the baseline from the thermogram. 
		NOTE: This type of manipulation is recommended only after multiple thermograms are collected, so the experimenter knows what is real data and what is an artifact, as adjusting the baseline and Integration Regions may drastically affect the data.</li>
		<li>Navigate to the Modeling window to begin fitting the data where the analysis software will show the integrated and concentration-normalized data for each injection.</li>
		<li>Left-click on the datum of the first injection to remove it from the fitting algorithm. 
		NOTE: This is common since there will be minor mixing between the titrant and sample cell solution leading to inexact molar dispensing of the first injection.</li>
		<li>In the Models section, select Blank (constant) in the Style dropdown menu, which is based on the final injection enthalpy and will be subtracted from every data point, accounting for the heat of dilution. In addition, select Independent (or the best model for the system) in the second Style dropdown menu to fit the data. 
		NOTE: For standard 1:1 binding interactions, the most common model is Independent.</li>
		<li>Fit the data using the two models by pressing the green play button with a &#931;. 
		NOTE: Another software to process ITC data is SEDPHAT24.</li>
		<li>Account for all competing equilibria in post hoc analysis previously reported by Grossoehme et al.20.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Waldron, K. J., Robinson, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+bacterial+cells+ensure+that+metalloproteins+get+the+correct+metal.">How do bacterial cells ensure that metalloproteins get the correct metal.</a> Nature Reviews Microbiology. 7 (1), 25-35 (2009).</li><li>Puig, S., Thiele, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+copper+uptake+and+distribution.">Molecular mechanisms of copper uptake and distribution.</a> Current Opinion in Chemical Biology. 6 (2), 171-180 (2002).</li><li>Huffman, D. L., O'Halloran, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function,+structure,+and+mechanism+of+intracellular+copper+trafficking+proteins.">Function, structure, and mechanism of intracellular copper trafficking proteins.</a> Annual Review of Biochemistry. 70 (1), 677-701 (2001).</li><li>Kaplan, J. H., Lutsenko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+transport+in+mammalian+cells:+Special+care+for+a+metal+with+special+needs.">Copper transport in mammalian cells: Special care for a metal with special needs.</a> Journal of Biological Chemistry. 284 (38), 25461-25465 (2009).</li><li>Makowska, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+binding+of+Cu(II)+ions+to+a+fragment+of+the+A&#946;+(1-42)+polypeptide+using+fluorescence+spectroscopy,+isothermal+titration+calorimetry+and+molecular+dynamics+simulations.">Probing the binding of Cu(II) ions to a fragment of the A&#946; (1-42) polypeptide using fluorescence spectroscopy, isothermal titration calorimetry and molecular dynamics simulations.</a> Biophysical Chemistry. 216, 44-50 (2016).</li><li>Gaggelli, E., D'Amelio, N., Valensin, D., Valensin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1H+NMR+studies+of+copper+binding+by+histidine-containing+peptides.">1H NMR studies of copper binding by histidine-containing peptides.</a> Magnetic Resonance in Chemistry. 41 (10), 877-883 (2003).</li><li>Garc&#237;a, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+and+electronic+structure+studies+of+copper(II)+binding+to+His111+in+the+human+prion+protein+fragment+106&#8722;115:+evaluating+the+role+of+protons+and+methionine+residues.">Spectroscopic and electronic structure studies of copper(II) binding to His111 in the human prion protein fragment 106&#8722;115: evaluating the role of protons and methionine residues.</a> Inorganic Chemistry. 50 (5), 1956-1972 (2011).</li><li>Jakab, N. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+histidine+containing+peptides+for+better+understanding+of+their+coordination+mode+toward+copper(II)+by+CD+spectroscopy.">Design of histidine containing peptides for better understanding of their coordination mode toward copper(II) by CD spectroscopy.</a> Journal of Inorganic Biochemistry. 101 (10), 1376-1385 (2007).</li><li>Keltner, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+characterization+and+activity+of+zinc-activated+proinsulin+C-peptide+and+C-peptide+mutants.">Mass spectrometric characterization and activity of zinc-activated proinsulin C-peptide and C-peptide mutants.</a> Analyst. 135 (2), 278-288 (2010).</li><li>Whittal, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Copper+binding+to+octarepeat+peptides+of+the+prion+protein+monitored+by+mass+spectrometry.">a Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry.</a> Protein science: a publication of the Protein Society. 9 (2), 332-343 (2000).</li><li>&#379;amoj&#263;, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pentapeptide+with+tyrosine+moiety+as+fluorescent+chemosensor+for+selective+nanomolar-level+detection+of+copper(II)+ions.">A pentapeptide with tyrosine moiety as fluorescent chemosensor for selective nanomolar-level detection of copper(II) ions.</a> International Journal of Molecular Sciences. 21 (3), 1-16 (2020).</li><li>Beuning, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+interpeptidic+Cu+II+exchange+rate+constants+of+Cu+II+-amyloid-&#946;+complexes+to+small+peptide+motifs+by+tryptophan+fluorescence+quenching.">Measurement of interpeptidic Cu II exchange rate constants of Cu II -amyloid-&#946; complexes to small peptide motifs by tryptophan fluorescence quenching.</a> Inorganic Chemistry. 60 (11), 7650-7659 (2021).</li><li>Makowska, J., &#379;amoj&#263;, K., Wyrzykowski, D., Wiczk, W., Chmurzy&#324;ski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper(II)+complexation+by+fragment+of+central+part+of+FBP28+protein+from+Mus+musculus.">Copper(II) complexation by fragment of central part of FBP28 protein from Mus musculus.</a> Biophysical Chemistry. 241, 55-60 (2018).</li><li>Stevenson, M. J., Farran, I. C., Uyeda, K. S., San Juan, J. A., Heffern, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+metal+effects+on+C-peptide+structure+and+internalization.">Analysis of metal effects on C-peptide structure and internalization.</a> ChemBioChem. 20 (19), 2447-2453 (2019).</li><li>Stevenson, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+a+copper+binding+site+in+proinsulin+C-peptide+and+its+implications+for+metal-modulated+activity.">Elucidation of a copper binding site in proinsulin C-peptide and its implications for metal-modulated activity.</a> Inorganic Chemistry. 59 (13), 9339-9349 (2020).</li><li>Magyar, J. S., Godwin, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectropotentiometric+analysis+of+metal+binding+to+structural+zinc-binding+sites:+Accounting+quantitatively+for+pH+and+metal+ion+buffering+effects.">Spectropotentiometric analysis of metal binding to structural zinc-binding sites: Accounting quantitatively for pH and metal ion buffering effects.</a> Analytical Biochemistry. 320, 39-54 (2003).</li><li>Ferreira, C. M. H., Pinto, I. S. S., Soares, E. V., Soares, H. M. V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(Un)suitability+of+the+use+of+pH+buffers+in+biological,+biochemical+and+environmental+studies+and+their+interaction+with+metal+ions+-+a+review.">(Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions - a review.</a> RSC Advances. 5 (39), 30989-31003 (2015).</li><li>Sigel, H., Martin, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+properties+of+the+amide+bond.+stability+and+structure+of+metal+ion+complexes+of+peptides+and+related+ligands.">Coordinating properties of the amide bond. stability and structure of metal ion complexes of peptides and related ligands.</a> Chemical Reviews. 82 (4), 385-426 (1982).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteins+containing+cytochrome,+iron-sulfur,+or+copper+redox+centers.">Metalloproteins containing cytochrome, iron-sulfur, or copper redox centers.</a> Chemical Reviews. 114 (8), 4366 (2014).</li><li>Grossoehme, N. E., Spuches, A. M., Wilcox, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+isothermal+titration+calorimetry+in+bioinorganic+chemistry.">Application of isothermal titration calorimetry in bioinorganic chemistry.</a> Journal of Biological Inorganic Chemistry. 15 (8), 1183-1191 (2010).</li><li>Miessler, G. L., Fischer, P. J., Tarr, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inorganic Chemistry. , (2014).</li><li>Walger, E., Marlin, N., Molton, F., Mortha, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+direct+red+81+dye/copper(II)-phenanthroline+system.">Study of the direct red 81 dye/copper(II)-phenanthroline system.</a> Molecules. 23 (2), 1-23 (2018).</li><li>Kocy&#322;a, A., Pomorski, A., Kr&#281;&#380;el, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molar+absorption+coefficients+and+stability+constants+of+Zincon+metal+complexes+for+determination+of+metal+ions+and+bioinorganic+applications.">Molar absorption coefficients and stability constants of Zincon metal complexes for determination of metal ions and bioinorganic applications.</a> Journal of Inorganic Biochemistry. 176, 53-65 (2017).</li><li>Zhao, H., Piszczek, G., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEDPHAT+-+A+platform+for+global+ITC+analysis+and+global+multi-method+analysis+of+molecular+interactions.">SEDPHAT - A platform for global ITC analysis and global multi-method analysis of molecular interactions.</a> Methods. 76, 137-148 (2015).</li><li>NIST. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Critically Selected Stability Constants of Metal Complexes, Version 8.0. , (2004).</li><li>Riener, C. K., Kada, G., Gruber, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+measurement+of+protein+sulfhydryls+with+Ellman's+reagent+and+with+4,4&#8242;-dithiodipyridine.">Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4&#8242;-dithiodipyridine.</a> Analytical and Bioanalytical Chemistry. 373 (4-5), 266-276 (2002).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li></ol>

The authors declare no competing interests.

This article provides a robust method for quantifying the affinity and thermodynamics of Cu(II) binding to peptides. Complexes with Cu(II) are ideally suited to monitor the d-d absorption band at the metal site due to its d9 electron configuration. Although the extinction coefficient is small, thus requiring larger concentrations of the complex to yield a reliable signal, titrations of Cu(II) into peptide can quickly provide insight into the binding stoichiometry and approximate binding affinity. However, it c...

Biology has evolved to utilize the diverse chemistry of metal ions needed for life to adapt and survive in its surrounding environment. An estimated 25%-50% of proteins use metal ions for structure and function1. The particular role and redox state of the metal ion is directly related to the composition and geometry of the biological ligands that coordinate it. In addition, redox-active metal ions such as Cu(II) must be tightly regulated lest they interact with oxidizing agents via Fenton-like chemistry to form reactive oxygen species (ROS)2,3,4. Understanding the binding modes and affinity that drive its biochemistry should help elucidate the biological role of the metal ion.
Many techniques are used to study the binding interactions of metals and peptides. These are mostly spectroscopic techniques but also include computer simulations using molecular dynamics, as seen through Cu(II) interactions with a fragment of amyloid beta (A&#946;)5. A widely used spectroscopic technique that is accessible to many universities is nuclear magnetic resonance (NMR). By using the paramagnetic nature of Cu(II), Gaggelli et al. were able to show where the metal ion binds on a petide through relaxation of nearby nuclei6. Electron paramagnetic resonance (EPR) may also be utilized to probe the location and mode of the paramagnetic metal ion binding7. Other spectroscopic techniques such as circular dichroism (CD) can describe the coordination about Cu(II) in systems such as tripeptide systems8, and mass spectrometry can show stoichiometry and to which residues the metal ion is coordinated through fragmentation patterns9,10.
Some of these techniques, such as NMR, are label-free but require large concentrations of peptide, posing challenges for study. Another common technique called fluorescence spectroscopy has been utilized to relate the position of a tyrosine or tryptophan with quenching from a Cu(II)11,12. Similarly, this technique can show structural changes as a result of Cu(II) binding13. However, challenges with these metal-peptide binding studies are that they probe chromophoric amino acids such as tyrosine which not all systems have, that the metal ion binds under a classical model, and that the technique may not be conducive under physiological conditions. Indeed, several peptides are emerging that do not contain such chromophoric amino acids or bind under classical models, precluding the use of these techniques14,15. This article details approaches for assessing binding properties in these scenarios under physiologically relevant conditions.
Biological ligands may adopt different protonation states that can affect metal ion binding such as the imidazole ring on histidine. If pH is not consistently maintained, the results can be convoluted or conflicting. For this reason, buffers are an essential component in the study of metal-protein/peptide interactions. However, many buffers have been shown to favorably interact with metal ions16,17. In addition to competing with the biological molecule of interest, the buffer may have similar coordinating atoms that may be difficult to distinguish from the coordinating atoms of the peptide or protein. In this study, the focus is on electronic absorption spectroscopy and isothermal titration calorimetry (ITC) as two complementary techniques for studying Cu(II)-peptide interactions, with special considerations concerning buffer choice.
Electronic absorption spectroscopy is a rapid, widely accessible technique for studying metal-binding interactions. Irradiation with light in the ultraviolet (UV) or visible wavelengths can lead to absorption of metal-centered d-d bands, which provide valuable information on ligand classification, metal geometries, and apparent binding affinities18,19. For these complexes, direct titrations of metal ions into protein or peptide solutions can quantify binding stoichiometries and apparent binding affinities. In some cases, such as d5 or d10 electron configurations, the complex does not absorb light (i.e., is spectroscopically silent). In these spectroscopically silent transition metal complexes, these limitations can be circumvented by using a competing ligand that, upon coordinating to the metal ion, yields detectable charge transfer bands. In either case, this approach is limited to quantifying only stoichiometry and apparent binding affinity, and no insight into binding enthalpy is provided without approximations.
Complementing information obtained from electronic absorption spectroscopy, ITC is an attractive technique for direct and rigorous quantification of the binding enthalpy20. ITC directly measures the heat released or consumed during a binding event and, since the titration takes place at constant pressure, the heat measured is the enthalpy of all equilibria (&#916;HITC). In addition, the stoichiometry of the binding event (n) and the apparent binding affinity (KITC) are quantified. From these parameters, the free energy (&#916;GITC) and entropy (&#916;SITC) are determined, providing a thermodynamic snapshot of the binding event. As it does not rely on light absorption, ITC is an ideal technique for spectroscopically silent species, for example, d5 or d10 metal ion complexes. However, since calorimetry measures heat, any unmatched buffer systems and unaccounted-for equilibria may adversely affect the analysis to accurately determine the metal ion binding thermodynamics, and great care must be taken to address these factors20. If performed with the appropriate rigor, ITC is a robust technique for determining the thermodynamics of metal-protein/peptide complexes.
Here, a chromophorically silent copper-binding peptide, C-peptide, is used to demonstrate the complementary use of the two techniques. C-peptide is a 31 residue cleavage product (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) formed during insulin maturation; it lacks chromophoric residues but has been shown to bind Cu(II) with physiologically relevant affinity14,15. The Cu(II) binding site consists of the side chains of a glutamate and an aspartate as well as the N-terminus of the peptide14,15. These coordinating atoms closely resemble those of many commonly used buffered systems. Here, the tandem use of the d-d and charge transfer bands in electronic absorption spectroscopy and ITC in quantifying the Cu(II) binding thermodynamics to C-peptide is shown. The approach from studying Cu(II) binding to C-peptide can be applied to other metal ions and protein/peptide systems.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,10-phenanthroline</td>
			<td>Sigma Aldrich</td>
			<td>131377-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>bis-Tris buffer</td>
			<td>Fisher</td>
			<td>BP301-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle-top 0.45 micron membrane</td>
			<td>Nalgene</td>
			<td>296-4545</td>
			<td>Any filtration system that removes the resin without introducing contaminants is acceptable</td>
		</tr>
		<tr>
			<td>Copper(II) chloride</td>
			<td>Alfa Aesar</td>
			<td>12458</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic absorption spectrophotometer</td>
			<td>Varian</td>
			<td>Cary 5000</td>
			<td>Another suitable sensitive spectrophotometer is acceptable</td>
		</tr>
		<tr>
			<td>high affinity resin</td>
			<td>Sigma Aldrich</td>
			<td>C7901-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Isothermal titration calorimeter (ITC)</td>
			<td>TA Instruments</td>
			<td>Nano ITC Low Volume</td>
			<td></td>
		</tr>
		<tr>
			<td>ITC analysis software</td>
			<td>TA Instruments</td>
			<td>NanoAnalyze</td>
			<td>SEDPHAT (Methods. 2015, 76: 137&#8211;148) may also be used</td>
		</tr>
		<tr>
			<td>ITC software</td>
			<td>TA Instruments</td>
			<td>ITCRun</td>
			<td></td>
		</tr>
		<tr>
			<td>light-duty delicate wiper</td>
			<td>Kimwipe</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>loading syringe</td>
			<td>Hamilton</td>
			<td>Syr 500 uL, 1750 TLL-SAL</td>
			<td></td>
		</tr>
		<tr>
			<td>matched cuvettes</td>
			<td>Starna Cells, Inc</td>
			<td>16.100-Q-10/Z20</td>
			<td>Ensure that the window for the small volume cuvette matches the beam height of the spectrophotometer</td>
		</tr>
		<tr>
			<td>MOPS buffer</td>
			<td>Alfa Aesar</td>
			<td>A12914</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer software</td>
			<td>Cary</td>
			<td>WinUV Scan</td>
			<td></td>
		</tr>
		<tr>
			<td>spreadsheet program</td>
			<td>Microsoft</td>
			<td>Excel</td>
			<td>Any suitable spreadsheet program will work</td>
		</tr>
		<tr>
			<td>titration syringe</td>
			<td>TA Instruments</td>
			<td>5346</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrapure water</td>
			<td>Millipore Sigma</td>
			<td>Milli-Q</td>
			<td>Any water is okay as long as &#62;18 M&#937; resistance</td>
		</tr>
	</tbody>
</table>

quantifying the binding interactions between cu(ii) and peptide residues in the presence and absence of chromophores

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been reported to directly bind to a variety of peptides and play both necessary and pathological roles ranging from mediating structure to electron transfer properties to imparting catalytic function. Quantifying the binding affinity and thermodynamics of these Cu(II)-peptide complexes in vitro provides insight into the thermodynamic driving force of binding, potential competitions between different metal ions for the peptide or between different peptides for Cu(II), and the prevalence of the Cu(II)-peptide complex in vivo. However, quantifying the binding thermodynamics can be challenging due to a myriad of factors, including accounting for all competing equilibria within a titration experiment, especially in cases where there are a lack of discrete spectroscopic handles representing the peptide, the d-block metal ion, and their interactions.
Here, a robust set of experiments is provided for the accurate quantification of Cu(II)-peptide thermodynamics. This article focuses on the use of electronic absorption spectroscopy in the presence and absence of chromophoric ligands to provide the needed spectroscopic handle on Cu(II) and the use of label-free isothermal titration calorimetry. In both experimental techniques, a process is described to account for all competing equilibria. While the focus of this article is on Cu(II), the described set of experiments can apply beyond Cu(II)-peptide interactions, and provide a framework for accurate quantification of other metal-peptide systems under physiologically relevant conditions.

The goal was to quantify and corroborate the thermodynamics of Cu(II) binding to C-peptide using the complementary techniques of electronic absorption spectroscopy and ITC. Due to the robust nature of electronic absorption spectroscopy, a direct titration of Cu(II) into 300 &#181;M C-peptide was performed (Figure 1). Addition of 150 &#181;M of Cu(II) caused an immediate increase in the band at 600 nm, attributed to the d-d band of Cu(II), and continued to increase until 300 &#181;M Cu(II) wa...

Watch this Scientific Journal Video about Quantifying the Binding Interactions Between CuII and Peptide Residues in the Presence and Absence of Chromophores at JoVE.com

Quantifying the Binding Interactions Between CuII and Peptide Residues in the Presence and Absence of Chromophores

This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been ...

quantifying-binding-interactions-between-cu-ii-peptide-residues

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

2.3K Views.  University of San Francisco. This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

Video: Quantifying the Binding Interactions Between CuII and Peptide Residues in the Presence and Absence of Chromophores

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

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SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer