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Two graphical rules for calculating the reaction rates of steady-state enzyme-catalysed systems are presented. According to Rule 1, the number of non-zero terms can be easily calculated, which is useful to check the results and avoid mistakes. Rule 2 provides a new graphical method, which will be much more effective than the existing methods in dealing with complicated problems. In addition, during the process of deriving Rule 1, the mathematical principles of the Wong-Hanes structural rule have been naturally obtained too.

The most abundant chlorophyll-binding complex in plants is the intrinsic membrane protein light-harvesting complex II (LHC II). LHC II acts as a light-harvesting antenna and has an important role in the distribution of absorbed energy between the two photosystems of photosynthesis. We used spectroscopic techniques to study a synthetic peptide with identical sequence to the LHC IIb N terminus found in pea, with and without the phosphorylated Thr at the 5th amino acid residue, and to study both forms of the native full-length protein. Our results show that the N terminus of LHC II changes structure upon phosphorylation and that the structural change resembles that of rabbit glycogen phosphorylase, one of the few phosphoproteins where both phosphorylated and non-phosphorylated structures have been solved. Our results indicate that phosphorylation of membrane proteins may regulate their function through structural protein-protein interactions in surface-exposed domains.

A complete analysis of calbindin D9k by two-dimensional 1H nuclear magnetic resonance spectroscopy has established the existence of two conformations for the folded protein in solution. Well-resolved major and minor resonances in a ratio of 3:1 are observed throughout the 1H NMR spectrum. Two-dimensional exchange experiments show that the major and minor species are related by an equilibrium process. Analysis of short proton-proton distances along the peptide backbone, identified by two-dimensional nuclear Overhauser effect spectroscopy, provides unambiguous evidence that the two forms of the folded protein differ only in the isomerization state of the peptide bond between Gly-42 and Pro-43. Cis-trans isomerism of Pro-43 is thereby directly identified as the cause of multiple conformations for the folded protein in solution. In addition, when Pro-43 is mutated to a glycine residue there is no indication of multiple conformations. These results provide evidence for the possibility of conformational heterogeneity in the native state of globular proteins.

The interaction between a variety of polyamines, both naturally occurring and synthetic, and calf thymus DNA has been studied using 23Na NMR. The relaxation behaviour of 23Na reflects the extent of interaction of Na+ with DNA phosphate groups and therefore the extent of charge neutralisation of DNA phosphate groups (P) by polyamine amino and imino groups (N) in solutions of DNa, polyamine and Na+. The studies reveal that whereas spermine and spermidine are capable of expelling nearly all of the Na+ ions from DNA at N/P approximately 1, diamines such as putrescine and homologues of spermine and spermidine are capable of neutralising only roughly 50% of DNA phosphates. The results provide a challenge to current models of DNA-polyamine interactions.

The structure of skeletal troponin C (sTnC) comprises two independently folded domains connected by an alpha-helical loop (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659). Both the NH2-terminal domain TR1C (9-84) and the COOH-terminal domain TR2C (89-159) can be obtained by limited tryptic cleavage. Here we report on proton and cadmium-113 NMR studies of Ca2+, Mg2+, and Cd2+ binding to these two separated domains. Our studies indicate that both halves retain a structure in the apo- and Ca2+-forms which resembles that of the intact protein. The events accompanying Ca2+ binding to these fragments are consistent with a biphasic binding pattern for sTnC, where the two sites in the COOH-terminal half are filled before those in the NH2-terminal half. Mg2+ only binds strongly to the two calcium-binding sites in the COOH-terminal half. The binding of this metal ion gives rise to a similar conformation for TR2C as that obtained with Ca2+. Whereas the binding of Ca2+ to the COOH-terminal part of sTnC takes place in a positive cooperative manner, the binding of this metal ion to TR2C occurs sequentially. Nevertheless, both Ca2+ ions bound to TR2C are in slow exchange (koff less than 10s-1). The binding of Mg24 both to sTnC and TR2C follows a sequential pattern with one site in slow exchange (koff less than 20 s-1) and one site in fast exchange (koff greater than 800 s-1). Binding of Cd2+ to TR2C occurs in a positive cooperative manner and results in the same conformation as observed with Ca2+. Cadmium-113 NMR spectra obtained at 5 degrees C confirm that two strong Cd2+-binding sites are localized in the COOH-terminal half and two weaker ones in the NH2-terminal half. It is concluded that these two proteolytic fragments comprise reasonable structural models for intact sTnC.

The accommodation of Mg2+ in the N-terminal domain of calmodulin was followed through amide 1H and 15N chemical shifts and line widths in heteronuclear single-quantum coherence spectroscopy NMR spectra. Mg2+ binds sequentially to the two Ca2+-binding loops in this domain, with affinities such that nearly half of the loops would be occupied by Mg2+ in resting eukaryotic cells. Mg2+ binding seems to occur without ligation to the residue in the 12th loop position, previously proven largely responsible for the major rearrangements induced by binding of the larger Ca2+. Consequently, smaller Mg2+-induced structural changes are indicated throughout the protein. The two Ca2+-binding loops have different Mg2+ binding characteristics. Ligands in the N-terminal loop I are better positioned for cation binding, resulting in higher affinity and slower binding kinetics compared with the C-terminal loop II (koff = 380 +/- 40 s-1 compared with approximately 10,000 s-1 at 25 degreesC). The Mg2+-saturated loop II undergoes conformational exchange on the 100-microseconds time scale. Available data suggest that this exchange occurs between a conformation providing a ligand geometry optimized for Mg2+ binding and a conformation more similar to that of the empty loop.