2004;279:31050C31057. the emission maximum and intensity measured. A comparison of the results from the two units of probes discloses that sites with most considerable cross-linking are also associated with the best changes in fluorescence. Titration of four different DAN-2 variants (351, 356, 365 and 367) with 2 gave a Kd of 0.4 M for subunit conversation. Disruption of the conversation of 2DAN-2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high throughput screen for inhibitors of subunit interactions. Ribonucleotide reductases GSK-923295 (RNRs) catalyze the conversion of nucleoside-5-di or triphosphates (NDPs or NTPs) to deoxynucleoside-5-di- or tri-phosphates (dNDPs GSK-923295 or dNTPs) in all organisms, and thus provide the essential precursors for DNA replication and repair(1, 2). In GSK-923295 2 (761 residues) in complex with a peptide composed of residues 356-375 of the C-terminus of 2 (2 has 375 amino acids) was crystallized and solved. In this structure, residues 358-375 of the peptide were visible(8, 13). The binding mode of this peptide was proposed to be indicative of how C-terminal tail of 2 binds to 2. Many structures of 2 are also available. In all cases, only residues 1-340 are visible(11, 12), with the remaining 35 residues, including 358-375, being disordered. Thus, no structural information is yet available about residues 341-357 of 2 and their functions in mediating subunit interactions. Based on the individual structures of 2 and 2, Eklund and coworkers generated a docking model of a 1:1 complex of 22 using shape complimentarily and charge compatibility(8). The model is the basis for the 35 ? distance proposed between the essential Y? (Y122) in 2 and the active site cysteine (C439) in 2. Recent pulsed electron-electron double resonance experiments support this long distance and the docking model(14, 15). An independent validation of the model through identification of 22 conversation sites is required to increase our understanding of this unique long-range radical transfer pathway and its control by GSK-923295 allosteric effectors. is an obligate dimer (2), while is an equilibrium mixture of monomer and dimer (2) with the dimer predominating in the presence of nucleotide (3) Interactions between 2 and 2 in all class I RNRs thus far examined are weak and largely dependent on the C-terminal 10-20 amino acids of 2 (16-18). The poor conversation, the potential changes in this conversation in the presence of substrates and/or allosteric effectors that bind to 2, and the consequences of these changes on enzymatic activity have prompted a number of studies to determine the Kds for subunit conversation (19-21). Early studies used GSK-923295 sucrose gradient ultracentrifugation and showed that allosteric effectors are required in the gradient for detection of 22 complex formation(4). Inhibition studies with 2, 2, CDP and ATP monitoring rate of dNDP formation using inactive 2 (Y122F) or using peptides of varying length corresponding to C-terminus of 2, revealed a Km of 0.2 M for 2 and 2 (18, 21). Recently, surface plasmon resonance techniques were examined in an effort to determine the influence of allosteric effectors and/or substrates on subunit affinity (20). Regrettably, technical problems in attaching 2 to the sensor SF1 chips have limited data interpretation. Thus, there is a gap in our quantitative understanding of complex formation between the subunits. Such information may well be important for the development of a quantitative description of allosteric regulation in class Ia RNRs in general and RNR specifically. In the present paper, development of a method to probe subunit interactions is reported in which all surface reactive cysteines in 2 are removed and a single cysteine has been incorporated site-specifically into 15 positions in its C-terminal tail. Each cysteine mutant has been modified with the photo cross-linker benzophenone (BP).