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A combination of differential titration calorimetry and differential scanning calorimetry was used to study the effect of disulfide bond cleavage and reaction with iodoacetamide of ribonuclease T, on both the binding of nucleotides and the thermal stability of the free enzyme species. Although guanosine monophosphates still bind to the active site of the modified protein the transition temperature of unfolding and thle transition enthalpy decrease drastically indicating a relatively loose structure. The calorimetric data presented in this study suggest a cooperative linkage between the site of the disulfide bonds, the ligand-binding site, and the general thermodynamic stability of the enzyme.Keywords. Microcalorimetry ; disulfide bonds ; cooperativity ; ligand binding ; ribonuclease T,.The biological function of proteins depends to a high degree on a balance between rigidity and flexibility of the protein structure. In addition to internal van der Waals' interactions and hydrogen bonds disulfide bridges are an important stabilizing feature of proteins.Since the thermal stability is a limiting factor in producing proteins of industIia1 importance, many attempts have been made to introduce disulfide bridges in order to improve the yield of the fermentation process or decrease the loss induced by denaturation. Recent results have demonstrated that the improvement of protein stability by the introduction of disulfide bonds is relevant to their location in the protein. The most effective stabilizing disulfide bridges are those introduced into the hydrophobic core without any major disturbance of the general protein structure (Zhou et al., 1993).Microcalorimetry (Hinz, 1986) is a suitable method to provide information about structural stability, by means of the free energy of reaction, AG,, and structural flexibility in terms of the entropy of reaction, AS,. The directly measured calorimetric enthalpy, AH,, can be assigned essentially to van der Waals and electrostatic interactions.Due to the complexity of the entropic contributions (e.g. backbone flexibility, excluded volume of the reaction partners, watedprotein interactions, water-ordering effect), a quantitative interpretation of the macroscopic parameter AS, is often difficult. Moreover, recent theories suggest that in addition to conformational entropic effects, enthalpic and native-state effects of disulfide bridges occur and cannot be neglected. It is argued that cross-links, such as disulfide bridges, destabilize the folded structure entropically, but stabilize it enthalpically to a greater Correspondence 10 H. Ruterjans, Institut fur Biophysikalische F a : +49 69 5800 9632. Abbreviations. 2'CMP, cytidine 2'-monophosphate; 2'GMP, guanosine 2'monophosphate ; 3'GMP, guanosine 3'-monophosphate; DSC, differential scanning calorimetry ; DTC, differential titration calorimetry ; Cam, carboxyamidomethyl; Cm, carboxymethyl.Enzymes. Ribonuclease A (EC 3.1.27.5); ribonuclease T, (EC 3.1.27.3).Chemie, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany extent (Doig and Will...

Rabbit uteroglobin (rab-UG) is a 16-kDa homodimeric secretory protein with potent anti-inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1 H, 15 N and 13 C resonances of the oxidized rabbit uteroglobin, based on several twodimensional and three-dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild-type as well as of uniformly 15 N-labeled and 15 N/ 13 C-labeled samples of the recombinant protein. A complete assignment of 1 H, 15 N and 13 C resonances, the secondary-structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab-UG and with the solution structure of human uteroglobin (h-UG). h-UG and rab-UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab-UG and a mutant with all cysteine residues exchanged to serine residues are discussed.Keywords : protein structure determination; uteroglobin; solution structure ; dimer; triple-resonance 1 H,Even though several three-dimensional structures of uteroglobins from different vertebrate species are known and a presumable association with their specific ligands has been shown, the precise physiological function of this protein is still a matter of discussion [1]. In fact, the main effort of investigation seems to be the search for the endogenous ligand, in order to draw conclusions about the possible regulatory function.Until recently, structural studies did not give conclusive information about the in vivo ligand. Uteroglobin (UG), also called blastokinin [2], was first discovered in the lumen of the rabbit uterus [3], where it seems to play an important, but still not completely understood role in the early stages of pregnancy.UG is a secretory dimeric protein with a molecular mass of 15 800 Da, consisting of two identical subunits of 70 amino acids each, which are covalently linked in an antiparallel manner by two disulphide bridges. The N-terminal Cys3 of monomer A CSI, chemical shift index; DQF, double-quantum filtered ; DSS, 2,2-dimethyl-3-silapentane-5-sulfonic acid; GARP, globular optimized, alternating-phase rectangular pulses ; h, human; HSQC, heteronuclear single-quantum coherence ; NTA, nitrilotriacetic acid; PEP, preservation of equivalent pathways; PFG, pulse field gradients; PLA 2 , phospholipase A 2 ; rab, rabbit; REDAC, redundant dehedral angle constraint; TPPI, time proportional phase incrementation; UG, uteroglobin; WALTZ-16, wideband, alternating-phase long-power tec...