Is the binding of magnesium (II) to calmodulin significant? An investigation by magnesium-25 nuclear magnetic resonance

Previous reports on the interaction between calmodulin (CaM) and Mg2+ range from no binding to a binding constant of 10(4) M-1 [for a summary, see Cox, J. A., Comte, M., Malnoe, A., Berger, D., & Stein, E. A. (1984) Met. Ions Biol. Syst. 17, 215-273]. In order to resolve the controversy, we used 25Mg NMR to study the binding of Mg2+ to apo-CaM, CaM.Ca2(2)+ (in which sites III and IV are occupied by Ca2+), CaM.La2(3)+ (in which sites I and II are occupied by La3+), and the two tryptic fragments of calmodulin, TR1C (containing sites I and II of CaM) and TR2C (containing sites III and IV of CaM). In each system, a "titration set" and a "temperature set" were obtained, and the spectral data were analyzed by total band-shape analysis to calculate the association constant (Ka) and off-rate (koff). As in the case of Ca2+ binding, sites I and II and sites III and IV were treated as two sets of equivalent sites, and a Ca2+/Mg2+ competition experiment suggested that Mg2+ competes with Ca2+ for the same sites. For both CaM.Ca2(2)+ and TR1C, moderately large Ka (2000 and 3500 M-1, respectively) and moderate off-rates (koff = 2300 and 3000 s-1, respectively, at 25 degrees C) were observed. For both CaM.La2(3)+ and TR2C, binding of Mg2+ was weaker by a factor of ca. 10 (Ka = 300 and 200 M-1, respectively) while the off-rates were also moderate (koff = 3500 and 2200 s-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

A new species of Croton (Euphorbiaceae) from Somalia

Croton crispatus , sp. nov., from Acacia-Commiphora bushland on sand in central Somalia, is described and illustrated.     

Acacia ochracea (Leguminosae-Mimosoideae), a new species from Somalia

Acacia ochracea , a new species in the A. Senegal complex, is described and illustrated. It is a common and conspicuous tree over large areas in the western Bay Region and in the Gedo Region in SW Somalia.     

New or noteworthy species of Leguminosae in NE tropical Africa

The following new species are described: Chamaecrista dunensis (Somalia), Acacia densispina (Somalia), A. flagellaris (Somalia), Tephrosia karkarensis (Somalia), T. scopulata (Somalia), Indigofera ancistrocarpa (Kenya), I. curvirostrata (Ethiopia), I. hiranensis (Somalia), I. karinensis (Somalia), I. kucharii (Somalia), I. nugalensis (Somalia), I. pellucida (Kenya, Somalia), I. sabulosa (Ethiopia, Somalia), I. sessilis (Somalia), I. ienuirostris (Somalia), Rhynchosia megalocalyx (Somalia), Crotalaria allophylla (Somalia), C. intricata (Somalia), C. warfae (Somalia), and Coronilla somalensis (Somalia). 2n = 22 is reported in Tephrosia karkarensis. Cassia somalensis, Acacia puccioniana, Sophora somalensis , and Crotalaria sennii are reduced to synonyms of respectively Senna hookeriana, Dichrostachys kirkii, Millettia usaramensis and Crotalaria boranica subsp. trichocarpa . Thirty-seven taxa are reported from Somalia for the first time, including Ptycholobium biflorum , known previously only from Southern Africa. Crotalaria pieropoda , known previously only from Socotra, is reported from Somalia and Oman. Neptunia oleracea and Vatovaea pseudolablab are new generic records for respectively Ethiopia and Oman. The fruits of Indigofera boranica are described. Vigna somaliensis and an allied species are discussed. Rhynchosia gansole is lectotypified and its flowers and fruits are described.     

Notes on Convolvulus, Astripomoea, Ipomoea and Merremia (Convolvulaceae) from the Horn of Africa

The six new species Convolvulus scopulatus, a shrub from gypseous semidesert coastal plains in northern Somalia, Astripomoea procera, a woody climber in Acacia-Commiphora bushland on sand in south-central Somalia, Ipomoea hiranensis, a prostrate or climbing shrublet in Acacia-Commiphora bushland in rocky places on sandstone in south-central Somalia, Ipomoea galhareriana, a woody climber in bushland on sand in central Somalia, Ipomoea pogonantha, a woody climber in bushland usually on red soil over limestone in eastern and southern Ethiopia, southern Somalia and northern Kenya, and Ipomoea corrugata, a herb with trailing stems in bushland on sandy soil in south-central Somalia, are described, and illustrations are provided for most of them. The new combinations Ipomoea ovatolanceolata (= I. adenoides var. ovatolanceolata) and Merremia obtusa (= M. ampelophylla subsp. obtusa) are made, and a new synonymy is provided for M. ampelophylla. Merremia ellenbeckii is neotypified and M. lobata is placed in synonymy.