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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.
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.
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
1. Electronic absorption spectroscopy: direct titration with buffer competition
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 µM C-peptide was performed (Figure 1). Addition of 150 µM 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 µM Cu(II) wa.......
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.......
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).
....Name | Company | Catalog Number | Comments |
1,10-phenanthroline | Sigma Aldrich | 131377-25G | |
bis-Tris buffer | Fisher | BP301-100 | |
Bottle-top 0.45 micron membrane | Nalgene | 296-4545 | Any filtration system that removes the resin without introducing contaminants is acceptable |
Copper(II) chloride | Alfa Aesar | 12458 | |
EDTA | Sigma Aldrich | EDS-500G | |
Electronic absorption spectrophotometer | Varian | Cary 5000 | Another suitable sensitive spectrophotometer is acceptable |
high affinity resin | Sigma Aldrich | C7901-25G | |
Isothermal titration calorimeter (ITC) | TA Instruments | Nano ITC Low Volume | |
ITC analysis software | TA Instruments | NanoAnalyze | SEDPHAT (Methods. 2015, 76: 137–148) may also be used |
ITC software | TA Instruments | ITCRun | |
light-duty delicate wiper | Kimwipe | 34155 | |
loading syringe | Hamilton | Syr 500 uL, 1750 TLL-SAL | |
matched cuvettes | Starna Cells, Inc | 16.100-Q-10/Z20 | Ensure that the window for the small volume cuvette matches the beam height of the spectrophotometer |
MOPS buffer | Alfa Aesar | A12914 | |
spectrophotometer software | Cary | WinUV Scan | |
spreadsheet program | Microsoft | Excel | Any suitable spreadsheet program will work |
titration syringe | TA Instruments | 5346 | |
ultrapure water | Millipore Sigma | Milli-Q | Any water is okay as long as >18 MΩ resistance |
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