Torbjörn Drakenberg

Calcium-induced structural changes and domain autonomy in calmodulin.

We have determined the solution structures of the apo and (Ca2+)2 forms of the carboxy-terminal domain of calmodulin using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The results show that both forms adopt well-defined structures with essentially equal secondary structure. A comparison of the structures of the two forms shows that Ca2+ binding causes major rearrangements of the secondary structure elements with changes in inter-residue distances of up to 15 Å and exposure of the hydrophobic interior of the four-helix bundle. Comparisons with previously determined high-resolution X-ray structures and models of calmodulin indicate that this domain is structurally autonomous.

Complete high-density lipoproteins in nanoparticle corona

In a biological environment, nanoparticles immediately become covered by an evolving corona of biomolecules, which gives a biological identity to the nanoparticle and determines its biological impact and fate. Previous efforts at describing the corona have concerned only its protein content. Here, for the first time, we show, using size exclusion chromatography, NMR, and pull‐down experiments, that copolymer nanoparticles bind cholesterol, triglycerides and phospholipids from human plasma, and that the binding reaches saturation. The lipid and protein binding patterns correspond closely with the composition of high‐density lipoprotein (HDL). By using fractionated lipoproteins, we show that HDL binds to copolymer nanoparticles with much higher specificity than other lipoproteins, probably mediated by apolipoprotein A‐I. Together with the previously identified protein binding patterns in the corona, our results imply that copolymer nanoparticles bind complete HDL complexes, and may be recognized by living systems as HDL complexes, opening up these transport pathways to nanoparticles. Apolipoproteins have been identified as binding to many other nanoparticles, suggesting that lipid and lipoprotein binding is a general feature of nanoparticles under physiological conditions.

Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase

The enzymes needed for galactomannan hydrolysis, i.e., beta-mannanase, alpha-galactosidase and beta-mannosidase, were produced by the filamentous fungus Aspergillus niger. The beta-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the beta-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger beta-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus beta-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.

Characterisation of 4-deoxy-β-l-threo-hex-4-enopyranosyluronic acid attached to xylan in pine kraft pulp and pulping liquor by 1H and 13C NMR spectroscopy

A new acidic sidegroup in xylans, from both kraft pulp and pulping liquor, was identified by NMR spectroscopy. Unmodified oligosaccharides from kraft pulp xylan were obtained by enzymatic hydrolysis with xylanase (Trichoderma reesei). The acidic oligosaccharides were separated from the natural forms on an anion exchange resin. The new acidic sidegroup was identified as 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid (hexenuronic acid) by 1H and 13C NMR spectroscopy. Hexenuronic acid is a beta-elimination product of 4-O-methylglucuronic acid and is formed during kraft pulping. HMBC and NOESY experiments showed that hexenuronic acid is attached beta-(1 --> 2) to xylose. The NOESY data further indicated that hexenuronic acid protrudes from the main xylan chain. The pKa values for hexenuronic acid (3.03) and 4-O-methylglucuronic acid (3.14) attached (1 --> 2) to xylose were determined from pH-dependent chemical shifts.

A 113Cd NMR study of calmodulin and its interaction with calcium, magnesium and trifluoperazine

Calcium has been long recognized as an important regulator of a variety of cellular events [l-5]. The detailed mechanism whereby calcium acts is still largely unknown. However, it has been demonstrated that calcium regulation of a number of enzyme systems is mediated by a low molecular weight, thermostable protein termed calmodulin. This protein was first described [4-7] as an activator of brain cyclic nudeotide phosphodiesterase. Cahnodulin has subsequently been found in tissues from various organs in both vertebrate, invertebrate and plant species [3,8,9,9a]. There is good evidence that calmoduliu represents an ubiquitous calcium regulatory protein whose primary sequence is highly conserved throughout all eucaryotic cells 133. Calmodulin (M, 16 700) consists of a single polypeptide chain whose amino acid composition is characterized by having a large number of acidic residues (glutamic and aspartic), a lack of cysteine and tryptophan, and the presence of one mole of the unusual amino acid trimethyllysine [lo]. Calmoduhn’s sequence is homologous with that of parvalbumin and skeletal troponin C (TnC) and like the latter its ammo acid sequence can be divided into 4 internally homologous domains, each of which has a potent~l calcium binding site [ 10 J. The calcium binding properties of calmodulin have been subject to several studies and while there is some inconsistency as regards the relative number of high and low affinity sites most studies indicate that calmodulin will bind 4 mol calciumlmol protein [ 1115 ] . Expe~ment~ evidence from a number of studies indicate that calcium binding to calmodulin is accompanied by pronounced changes in its solution conformation [ 1 l-14,16-22].

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.

1H, 13C, 35Cl, and 81Br NMR of aqueous hexadecyltrimethylammonium salt solutions: Solubilization, viscoelasticity, and counterion specificity

Abstract A combination of 1H, 13C, 35Cl, and 81Br NMR is used to investigate counterion specificity and solubilization behavior of hexaldecyltrimethylammonium micelles. The concentration dependence of the quadrupole relaxation of 81Br− and 35Cl− counterions in aqueous solutions of hexadecyltrimethylammonium bromide (CTAB) and chloride (CTAC), respectively, is found to be quite different and this can be referred to the formation of large elongated micelles in the former case but not in the latter. This striking difference is maintained in solutions containing both CTAB and CTAC. This is attributed to the coexistence of two types of micelles and a considerable specificity in the counterion binding, the Br− ions having a preference for the cylindrical and the Cl− ions for the globular micelles. For mixtures of CTAB with tetradecyltrimethylammonium chloride (TTAC), the same type of counterion specificity in 35Cl and 81Br NMR was observed as for mixtures of CTAB and CTAC. The 13C chemical shifts are closely the same for solutions of either CTAB, CTAC, or TTAC, but in mixtures of CTAB + TTAC or CTAC + TTAC there is a marked splitting of the ω-CH3 resonance into two peaks. This is referred to alkyl chain packing disturbances in mixed micelles of two amphiphiles with different alkyl chain lengths. 1H and 13C NMR were employed to study the solubilization site for viscoelastic solutions of CTAB and 1-methylnaphthalene. A highly variable effect along the amphiphile chain was observed for both 1H and 13C relaxation and shielding. It could be established that solubilization of 1-methylnaphthalene occurs toward the polar part of CTAB micelles, about seven methylenes being affected appreciably. Chain flexibility is found to be influenced in a complex way by solubilization.

Binding of Levosimendan, a Calcium Sensitizer, to Cardiac Troponin C

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

Phosphorylation controls the three-dimensional structure of plant light harvesting complex II.

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.

Non-equivalence of the CD and EF sites of muscular parvalbumins. A 113Cd NMR study

The tertiary structure of parvalbumins is well documented by the determination of atomic coordinates for the component ~14.25 from carp muscle by X-ray crystallography [l] . A significant feature of this structure is the presence of two calcium ions at sites named CD and EF respectively [2] . Six oxygen atoms are liganded to the central cation in an octahedral arrangement, with Ca . . .O distances ranging from 2.15-2.85 A [l] . The first site contains solely protein ligands: four carboxyl groups, a peptide bond carbonyl group and a serine hydroxyl group. The site EF also contains four carboxyl groups and a carbonyl group but the sixth ligand is provided by a water molecule [l] , This suggests that the parvalbumin sites CD and EF will have different physicochemical and functional properties. Studies of the binding of Ca(I1) by various techniques have shown that parvalbumins have two cationic sites with high affinity for calcium ions [3-51, but have not been able to distinguish between the two sites. It has also been shown that many other cations compete for the ionic sites of parvalbumins [6-91. Specific binding of Na(1) [lo] and Mg(II) [ 1 l] has been observed using “Na and “Mg NMR spectroscopy. Kinetic aspects of the binding of Ca(II) to parvalbumins have been investigated directly by 43Ca NMR [ 121. Although well adapted to charac-