The complex formation of glycyl-l-histidine, glycyl-l-histidylglycine, and glycylglycyl-l-histidine with copper (II) ion in aqueous solutions containing equimolar amounts of copper(II) and the respective ligand was investigated by potentiometric and visible spectrophotometric methods. The presence of the following copper (II) complex species was assumed: CuLH2+, CuX, and CunYnn− for glycyl-l-histidine-copper(II) and glycyl-l-histidylglycine–copper(II) systems, and CuLH2+, CuY−, and CuZ2− for glycylglycyl-l-histidine-copper(II) system, where LH is the neutral species of the ligand and LH=XH2=YH3=ZH4. The formation and ionization constants were obtained graphically, and the distribution of copper(II) among the complex species was calculated for each system. The results indicate that copper(II) ions are bound to the imidazole and carboxyl (or carbonyl) groups in CuLH2+ and to the amino, imidazole, and deprotonated amide groups in CuX, CuY−, and CuZ2−. The spectral blue shift accompanying a deprotonation in the region from a=4 to 5 in the glycyl-l-histidine and glycyl-l-histidylglycine copper(II) systems indicates the formation of a polymer complex CunYnn− in which the imidazole ring acts as a bridge to connect copper atoms, where a represents moles of KOH added per completely protonated ligand.

The cyclic AMP receptor protein (CRP) is a positive and negative regulatory protein for gene expression in Escherichia coli. The protein has been cleaved proteolytically to determine the relation between CRP structure and function. In the presence of sodium dodecyl sulfate (NaDodSO4), chymotrypsin dissects CRP into two stable fragments of molecular weight 9500 (9.5K) and 13 000 (13K). After removal of NaDodSO4, the two fragments are resolved by Bio-Rex 70 chromatography in 6 M urea. Analyses of the terminal amino acids released from each fragment and cyanogen bromide cleavage products indicate that the 9.5K fragment is amino proximal in CRP while the 13K fragment is carboxyl proximal. Notable features of amino acid composition are the relatively high amount of arginine and methionine in the 13K fragment and the retention in the 9.5K fragment of the two tryptophans present in the CRP subunit. Following isoelectric focusing in 8 M urea, the 9.5K fragment, 22.5K CRP, and 13K fragment migrate to pH 5.5, 8.3, and 10.3, respectively. While CRP is a cAMP-stimulated DNA binding protein, the 13K fragment binds to DNA in the presence and absence of cAMP. The 9.5K fragment associates to form dimers and decamers. These data are consonant with a model in which the DNA binding domain is present in the carboxyl proximal region of CRP while the amino proximal region contains the subunit-subunit interaction sites and much of the cAMP binding domain.

The binding of the cAMP receptor protein (CRP) to the portion of the lac promoter comprising the core of the CRP recognition sequence has been investigated. The effect of the binding of CRP to the symmetrical 22- and 28-base-pair operators was investigated by 1H NMR. The binding of cAMP*CRP to the 22mer DNA did not bring about any changes in the chemical shift values of the imino proton resonances of the DNA, but did cause selective line broadening of the imino proton resonances of specific base pairs (TA 4, GC 5). These base pairs are contained in the motif 5'(TGTGA)3', which is thought to be the region crucial to interaction with the CRP. The binding of cAMP*CRP to the 28mer DNA brought about large changes in the imino proton resonances that seem to be induced by DNA bending. Therefore it seems likely that CRP requires DNA longer than a 22mer to bend it. We also used a C-terminal protease-digested CRP (CRPcy) in a DNA-binding experiment. The binding of cAMP*CRPcy to the 28mer DNA did not induce bending. These results indicate that the C-terminal region of CRP participates in DNA binding and is important for DNA bending.

Equilibrium constants were determined in the complex formations of l-histidylgylcine (HG) and l-histidylglycylglycine (HGG) with copper(II) ion in aqueous solution by potentiometric titration, and the structures of the complexes were discussed based on the observations in the titration and infrared and electronic absorption spectra. In the acid region, a cationic chelate, in which copper(II) binds to nitrogen atoms of amino and imidazole groups, is formed. In the neutral region, deprotonation of the peptide amide group occurs to form a dimer, in which four coordination positions of copper(II) are occupied by nitrogen atoms of deprotonated amide and amino groups and an oxygen atom of carboxyl or carbonyl group in one molecule and a nitrogen atom of imidazole group of another molecule. In HGG–Cu(II) system, the dimer is decomposed in the alkaline region with further deprotonation of the second peptide amide group to form an anionic chelate as in the case of the glycylglycylglycine–Cu(II) complex.

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