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The complex formation of the N-terminal domain (headpiece) of the Escherichia coli lac repressor and a synthetic 14-base-pair lac operator fragment has been investigated by 1H NMR. Titration shifts in the imino-proton region of the DNA spectrum and in the aromatic region of the headpiece spectrum are examined in detail and interpreted where possible. The assignment of the resonances in the complex follows in part from the titration data and is completed by nuclear Overhauser measurements. The shift of the His-29 C-2 resonance has been used to assess the binding strength of the complex. Evidence is presented for the presence of a high-affinity site on the lac operator fragment (KD less than or equal to 2 X 10(-5) M), which shows features in common with one of the specific binding sites on the complete lac operator, and for the presence of a second, nonspecific binding site with lower affinity. The influence of this second site on the interpretation of the binding data is discussed.

The imino proton resonances of 15N labeled tRNA appear as asymmetric doublet signals, the asymmetry being dependent on the applied magnetic field strength. Assuming a tautomerism of the type N-H...N not equal to N...H-N in the base pairs the line shapes can be simulated. The most important parameters fitted in the simulation are the rate constants of the proton transfer and the mole fractions of either tautomeric state. The rate constants are of the order of 100s-1 and the mole fractions of the non dominant tautomer about 0.1 depending on the temperature and on the nature of the base pairing. The observations are attributed to a double proton transfer in the base pairs. The unexpectedly slow rates of the double proton transfer process may be connected with a concomitant conformational change of the duplex structure.

Individual haemoglobin I11 from Chironomus thummi thummi is a monomeric protein ( M , = 15500) which has recently been shown by us to be characterized by a simple Bohr effect. Three of the four histidines, the position of which in the polypeptide chain is known from sequence analysis, are distinguished by their titration characteristics, namely His-G2, His-GI9 and His-E5. The chemical shift of the C-2 proton resonances of the imidazole groups, which varies upon protonation of the histidine, has been used for the determination of the intrinsic p K values. His-F8, the proximally bound histidine, is not observable because its C-2 proton resonance is broadened.One of the three titratable histidines reveals a pK value which is varied in the range from 7.5 to 7.1, depending on the 6th ligand a t the haem iron. The relatively high pK value of this histidine and its abnormal value of the chemical shift of the C-2 proton resonance a t low pH indicate an interaction with a carboxylate group. This shift of the pK value caused by the binding of CO to reduced haemoglobin is in agreement with the interaction energy, calculated from the amplitude of the Bohr effect curve, and corresponds to 500 cal.I n this simple case the assignment of the C-2 proton resonances to the known histidines is possible considering the atomic model, described by Huber et al. [4]. The results indicate that His-G2 is the allosteric binding site; the remaining two detectable histidines are not involved in the allosteric interaction. The molecular mechanism of the Bohr effect in this haemoglobin is discussed.Contrary to the mammalian haemoglobins, the larval haemoglobins of the insect Chironomus thummi thummi are characterized by a simple, alkaline Bohr effect [I]. It is of special interest that two of these haemoglobins, namely individual haemoglobins I11 and IV, which are monomeric in the range of pH 5 to 10, also show this Bohr effect Recently we reported results of proton magnetic resonance studies of histidine residues of horse myoglobin [5]. I n the case of myoglobin nine of the eleven histidine C-2 proton magnetic resonances have been resolved and from the titration curves the intrinsic p K values of these histidines have been determined. I n this communication the results of a titration of the histidine residues of haemoglobin I11 are described. This haemoglobin has a molecular weight of 15800 [3] and a tertiary structure quite similar Abbreviations. NMR, nuclear magnetic resonance; Me,Si, tetramethylailane.to that of myoglobin [4]; however, only four histidines appear in the sequence : His-F8, His-G2, His-E5, and His-G19. This small number of histidines allows a more exact determination and assignment of the intrinsic pK values and of their possible variation in the case of conformational changes induced by ligand binding. Such a conformational change is re%ected by the Bohr effect.The Bohr effect describes a negative interaction between the 0,-binding site and proton-binding sites in the mammalian haemoglobins 16-81. The identification of the ...