Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II

Abstract

Human carbonic anhydrase (CA) is a well-studied, robust, mononuclear Zn-containing metalloprotein that serves as an excellent biological ligand system to study the thermodynamics associated with metal ion coordination chemistry in aqueous solution. The apo-form of human carbonic anhydrase II (CA) binds two equivalents of copper(II) with high affinity. The Cu2+ ions bind independently forming two non-coupled type-II copper centers in CA (CuA and CuB). However, the location and coordination mode of the CuA site in solution is unclear, compared to the CuB site that has been well characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we have identified an N-terminal Cu2+ binding location and collected information on the coordination mode of the CuA site in CA, which is consistent with a four to five coordinate N-terminal Cu2+ binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-ATCUN and copper(II)-beta-amyloid complexes. Additionally, we report a more detailed analysis of the thermodynamics associated with copper(II) binding to CA. Although we are still unable to fully deconvolute Cu2+ binding data to the high-affinity CuA site, we have derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu2+ binding to the CuB site of CA to be 2 × 109 and −17.4 kcal/mol, respectively.

High-Resolution Solid-State NMR Characterization of Ligand Binding to a Protein Immobilized in a Silica Matrix

“…86 The protein backbone assignment was obtained from the analysis of the solution NMR spectra acquired on hCAII and compared with literature data. 60,61 The final percentage of assignment was 88%, since residues 1-22 and some residues belonging to loops could not be identified in the spectra, in line with previous reports. 60,61…”

“…We have chosen hCAII because it has been already characterized by NMR. [57][58][59][60][61] hCAII is a very efficient enzyme that catalyzes the hydration of CO 2 to HCO 3 -, which would occur too slowly on the physiological timescale.…”

Global stability of an α-ketoglutarate-dependent dioxygenase (TauD) and its related complexes

“…These linked equilibria include changes in solvation and ionization states of the protein, metal ion, substrate and cofactor, and buffer components, in addition to protein aggregation [21,28–31]. Many of these linked equilibria have no impact on the secondary structure monitored by CD (mainly the cupin fold), however the contributions of these effects on the overall denaturation process are better captured by monitoring the change in heat capacity of the enzyme complex using the DSC.…”

Pull‐Down of Metalloproteins in Their Native States by Using Desthiobiotin‐Based Probes

“…Probe 4 can further be used to pull down CA in different metallation states including Mn 2+ ‐CA, Fe 3+ ‐CA, Co 2+ ‐CA, Ni 2+ ‐CA, Cd 2+ ‐CA, Cu 2+ ‐CA, and bis‐Cu 2+ ‐CA, which has Cu 2+ bound to the active site as well as to a secondary site. Of these, Mn 2+ ‐, Fe 3+ ‐, and Co 2+ ‐CAs were successfully captured (Figure C).…”