Name: quantifying the binding interactions between cu(ii) and peptide residues in the presence and absence of chromophores
Uploaded: 2022-04-05T15:00:00
Description: 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

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: ...

<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>

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 ...

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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

个性化肽阵列在器官移植 HLA 抗体检测中的研究

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

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

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

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 ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

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

测量细胞核与骨架间机械一体化的直接力探针

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

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

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

高速离心条件下 u937 细胞的细胞分裂

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