A cofactor approach to copper-dependent catalytic antibodies

A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, Lys(H93), that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2–5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl(2), or (ii) precoordination of 5 with CuCl(2) followed by conjugation with 38C2. The resulting 38C2–5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis–Menten kinetics [k(cat)(11) = 2.3 min(−1) and K(m)(11) 2.2 mM] with a rate enhancement [k(cat)(11)k(uncat)(11)] of 2.1 × 10(5). Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl(2)) gives a rate enhancement of 3.5 × 10(4) in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2

Interfacial metal and antibody recognition

The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca(2+), Ba(2+), or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with ≈1.5 kcal/mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition

Dynamics of Price Adjustment in Qualitatively Differentiated Markets in the U.S.: The Case of Organic and Conventional Apples

Production and consumption of conventional and organic apples in the U.S. have changed dramatically over the past two decades. Despite a drop in the conventional apple production, production of organic apples shows a double-digit growth. Demand for organic apples also continues to outpace growth in overall organic fruit sales. In this research, we investigate whether these changes have an impact on price adjustment dynamics between these two qualitatively differentiated products. We use a Markov-Switching Assymetric Vector Error Ccorrection model (MS-AVECM) and weekly U.S. national retail prices for the 2010-2015 period for three varieties of apples: Gala, Fuji, and Red Delicious. The MS-AVECM results indicate three Markov-Switching regimes can be defined for the three varieties during the study period. The results show there are different short-run price adjustment daynamics in each of these regimes and asymmetric price transmission behavior between organic and conventional apples. Result indicates that apple varity plays important role in the price relationship between organic and conventional apple. These findings have implications for farmers, wholesalers, retailers, policy makers, as well as consumers