Site‐Selective Photocleavage of Proteins by Uranyl Ions

Abstract

Dedicated to Professor J. C. Scaiano on the occasion of his 60th birthdayThe uranyl ion (UO 2 2+ ) has a high affinity for the phosphate groups of DNA/RNA, [1] and this affinity was exploited to photocleave these biopolymers at the uranyl-binding sites. [2][3][4][5] Earlier success in our laboratory with site-specific photocleavage of proteins by organic molecules [6] and metal complexes [7] led to the idea that photoactive [8] metal-oxo species such as UO 2 2+ should be capable of cleaving proteins with high selectivity. However, even though there is ample evidence to suggest that UO 2 2+ binds to proteins, [9, 10] it is not known if UO 2 2+ can photocleave the peptide backbone. It is also not known if binding of UO 2 2+ at specific sites would result in high selectivity for the protein cleavage. In addition, the ease with which UO 2 2+ can be directed to metal-binding sites on proteins and the activation of the cation at visible wavelengths indicated that this would be a promising approach for footprinting metalbinding sites. In this report, high-affinity binding of UO 2 2+ to proteins and their site-selective photocleavage, with visible light, is demonstrated for the first time.Serum albumin (SA) and transferrin are known to bind UO 2 2+ , [11,12] and these proteins therefore provided good initial targets to test the above approach. Quantitative data on the binding of UO 2 2+ to bovine SA (BSA), however, were lacking; these were obtained from calorimetric and spectroscopic studies. Isothermal titration calorimetry of the addition of a solution of UO 2 2+ (0.5 mm) to BSA (0.1 mm ; see Supporting Information) indicated exothermic binding (Figure 1). Best fits to the data required a two-site binding model [13] and indicated a high-affinity site (binding constant (K b ) of (1.6 AE 0.7) 10 7 m À1 ) with moderate exothermicity (binding enthalpy (DH) of (À4.7 AE 0.7) kcal mol À1 ) and a low-affinity site (K b = 2.8 AE 2.5 10 5 m À1 ) with weak exothermicity (DH = À(1.2AE0.8) kcal mol À1 ). Binding of UO 2 2+ to BSA was also supported by absorption, circular dichroism (CD), and fluorescence studies.The CD spectra, for example, of a mixture of BSA (1 mm) and UO 2 2+ in increasing concentrations (0-0.8 mm) showed the growth of new bands at 440 and 510 nm (Figure 2). These evidences established the binding of UO 2 2+ to BSA. Photocleavage of BSA by UO 2 2+ was achieved by irradiating a mixture of BSA and UO 2 2+ at 420 nm (Figure 3). Three distinct product bands at 35, 33 and 30 kDa were obtained in yields (11 AE 0.5), (4 AE 0.6), and (11 AE 1) % (lane 5), respectively. No photocleavage was noted without UO 2 2+ (lane 3) or without light (lane 4). Photocleavage was observed at wavelengths up to 460 nm, although the yields were lower (data not shown).To demonstrate the general nature of the photocleavage, other proteins such as human SA (HSA), porcine SA (PSA), glucose oxidase, and transferrin were also tested. These are cleaved with high selectivity (see Supporting Information). As negative controls, we also tested the ...

Size‐dependent dextran loading in protein nanotube with an interior wall of concanavalin A

“…(c)). It is known that the uranyl ion () binds strongly to the subdomain IIB of HSA . Therefore, the totally dark cylinders were attributed to the binding to the HSA components in the wall.…”

Uranyl Binding to Proteins and Structural-Functional Impacts

“…With excellent photocatalytic properties of uranyl ions [88], uranyl photocleavage has been observed not only for DNA/RNA [75,89], but also proteins, especially for those with phosphorylation, such as α-/β-casein and ovalbumin [90]. Duff and Kumar first demonstrated that UO 2 2+ was able to cleave a number of proteins, including BSA, HSA, porcine SA (PSA), glucose oxidase and transferrin, as induced by visible light irradiation (420-460 nm) [91]. Note that the cleavage site is highly selective, for example, the primary cleavage site on BSA is Val314-Cys315.…”

A Phosphorylation Tag for Uranyl Mediated Protein Purification and Photo Assisted Tag Removal

“…Despite the small difference in molecular mass (eleven amino acids), GFP0 and GFP28 show different mobility by SDS PAGE (lane 1, Figure 2A). It has previously been demonstrated that uranyl cleavage of non-modified bovine serum albumin (BSA) takes place upon irradiation [29]. Therefore, BSA was added to the E. coli protein extract as an internal non-phosphorylated control, and capture was done using uranyl-NTA agarose beads, which was found to be the most efficient matrix in terms of binding and elution of the phosphorylated protein (data not shown).…”