Barry A. Levine

The interaction of ions with phosphatidylcholine bilayers

The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in 2H2O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions. Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes. The apparent binding constant Kb varies as a function of the quantity of metal ion bound and as a function of the Cl− concentration. The apparent binding constant at “zero loading” is K0 = 1.25 · 104L2 · M−at 0.15 M KCl. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl− concentration. The interaction of lanthanides and divalent cations such as Ca2+ and Mg2+ is independent of pH in the pH range 3–7+ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is Cl− < Br− < NO3− < SCN− < I− < ClO4−. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions. The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca2+ and Mg2+ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO22+ showing the highest affinity.

Structural information from NMR secondary chemical shifts of peptide α C-H protons in proteins

The secondary chemical shift experienced by the1H-NMR resonances of the α C−H protons in proteins can be correlated with their backbone torsional angles ψ, which dictate the orientation of the α C−H proton to the adjacent carbonyl group. It is shown that α C−H protons present in β-sheet regions experience downfield secondary shifts , whereas those in α-helix regions experience upfield secondary shifts. The predictive use of this correlation in assignment studies is illustrated for the calcium-binding protein paravalbumin, for which a crystal structure is available, and troponin C, for which no crystallographic data are available.

Binding sites involved in the interaction of actin with the N-terminal region of dystrophin

Two actin‐binding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to defined regions of the polypeptide sequence. These are Actin‐Binding Site 1 (ABS1) located at residues 17–26 and Actin‐Binding Site 2 (ABS2) in the region of residues 128–156. Using defined fragments of the actin amino acid sequence, ABS1 has been shown to bind to actin in the region represented by residues 83–117 and ABS2 to the C‐terminal region represented by residues 350–375. These dystrophin‐binding sites lie on the exposed domain in the actin filament.

The interaction of actin with dystrophin.

Proton NMR spectroscopy of synthetic peptides corresponding to defined regions of human dystrophin has been employed to study the interaction with F‐actin. No evidence of interaction with a C‐terminal region corresponding to amino acid residues 3429–3440 was obtained. F‐actin restricted the mobility of residues 19–27 in a synthetic peptide corresponding to residues 10–32. This suggests that this is a site of F‐actin interaction in the intact dystrophin molecule. Identical sequences to that of residues 19—22 in dystrophin, namely Lys‐Thr‐Phe‐Thr are also present in the N‐terminal regions of the α‐actinins implying this is also a site of F‐actin interaction with α‐actinin.

A study of calmodulin and its interaction with trifluoperazine by high resolution 1H NMR spectroscopy

The antipsychotic drug trifluoperazine (TFP) is known to be an effective inhibitor of the calciumbinding activator protein, calmodulin (CaM). It has been shown that 2 mol TFP bind to 1 mol CaM with Kd lOA M [ 11. In [2] ‘13Cd NMR was used to study the effects of TFP binding to CaM on the cation binding sites of the protein. It was found that the binding of 2 mol drug to 1 mol protein affected all 4 cationbinding sites. We have been using high resolution ‘II NMR to study the conformation of calmodulin in solution under varying conditions. Our observations on the protein itself agree with those in [3]. Here we report the effects of drug binding on the ‘H NMR spectrum of calmodulin.

Calcium binding by troponin-C. A proton magnetic resonance study.

The conformational changes induced by the binding of Ca(II) to rabbit skeletal muscle troponin-C (TNC) have been followed by proton magnetic resonance spectroscopy. Ca(II)-free TNC (apo-TNC) contains definite ordered regions. Ca(II) titration of the high affinity sites (cf. Potter , Gergely, 1975) causes a large folding of the backbone, some of which involves refolding of an ordered region(s) and changes in several side-chains e.g. Glu, Asp and Phe. Titration of the low affinity sites does not alter the backbone but leads to changes among hydrophobic side-chains (one or more Val, Leu, Ile; two or more Phe, Glu and Asp) that define an ordered region(s) of apo-TNC. The rate constants for the conformation changes of the low and high affinity sites are approximately 10 s−1 and < 20 s−1, respectively. Final stages of the titration include a downfield shifted methyl group (likely Ile) and a Phe residue. The thermal stabilities of apo-TNC, TNC · Ca2(II) and native TNC were compared. It was concluded that Ca(II) binding by the two high affinity sites both directs and stabilizes much of the structure. The role of the changes of the low affinity sites, which are thought to activate contraction, are briefly discussed.

STRUCTURE AND FUNCTION OF X-PRO DIPEPTIDE REPEATS IN THE TONB PROTEINS OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI

The TonB protein is required for several outer membrane transport processes in bacteria. A short 33-residue peptide segment of TonB has been studied by 1H and 13C nuclear magnetic resonance spectroscopy. The sequence of this peptide segment contains multiple Glu-Pro and Lys-Pro dipeptide repeats that maintain rigid, elongated structures and flank a short connecting segment that adopts a beta-strand configuration. This TonB peptide is shown to interact specifically with the FhuA protein, the outer membrane receptor for ferrichrome-iron, providing the first direct evidence that the TonB protein interacts with outer membrane receptors. Interaction with the FhuA protein involves the extended structural element containing positively charged Lys-Pro repeats, and suggests a functional role for this segment of the TonB protein. As TonB is anchored in the cytoplasmic membrane the protein must, uniquely, span the periplasm. These data, together with studies described in the accompanying paper, suggest a model by which TonB serves to transduce conformational information over extended distances, from the cytoplasmic membrane to the outer membrane.

Sequence-imposed structural constraints in the TonB protein of E. coli

The solution conformation of a 33‐residue peptide segment derived from the TonB protein which is implicated in bacterial membrane transport processes, has been investigated using high‐resolution proton magnetic resonance techniques. This proline‐rich peptide possesses sequence‐imposed sections of elongated secondary structure that must be retained in the native protein configuration. These structural constraints provide elements of stiffness that imply a purely structural role for TonB and are relevant to the subcellular location and biological role of the protein. On the basis of these data we suggest that this protein spans the periplasmic space linking the inner and outer membrane components of TonB‐dependent transport systems.

The conformation of the polar group of lysophosphatidylcholine in H2O; conformational changes induced by polyvalent cations

The conformation of the polar group of egg lysophosphatidylcholine and 1-myristoyl-sn-glycer-3-phosphorylcholine present as micelles in aqueous solution has been studied using NMR methods. In the absence of polyvalent cations the preferred conformation derived from spin-spin coupling constants is similar, but not identical, to that of phosphatidylethanolamine in the crystal structure (cf. Hitchcock, P.B., Mason, R., Thomas, K.M. and Shipley, G.F. (1974) Proc. Natl. Acad. Sci. U.S. 71, 3036--3040). The presence of lanthanides induces a conformational change involving primarily the phosphorylcholine group, e.g. torsion angle alpha5 changes from an all gauche to an approximate trans disposition. The gauche leads to trans transitions observed with torsion angles alpha3 and alpha5 produce a more extended orientation of the polar group (relative to the hydrocarbon chain axis). In the presence of lanthanides the conformation of lysophosphatidylcholine is very similar to that of the diacyl phosphatidylcholines observed in fully hydrated bilayers (cf. Hauser, H., Phillips, M.C., Levine, B.A. and Williams, R.J.P. (1976) Nature 261, 390--394) with the P-N vector at an angle of about 45 degrees to the bilayer.

Multiple binding sites on the CH2 domain of IgG for mouse FcγR11

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (FcγR). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fcγ R. This domain was mapped for interaction with mouse Fcγ R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human FcγR1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237 → Ala, Asn 297 → Ala, and Glu 318 → Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse FcγR11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by FcγR11 of the P388D1 cell line.