Toshimichi Fujiwara

Molecular mobility of protein in lyophilized formulations linked to the molecular mobility of polymer excipients, as determined by high resolution 13C solid-state NMR.

AbstractPurpose. The mobility of protein molecules in lyophilized protein formulations was compared with that of excipient molecules based on the spin-lattice relaxation time (T1) of each molecule determined by high resolution 13C solid-state NMR. The relationship between molecular mobility and protein stability is discussed. Methods. Protein aggregation of lyophilized bovine serum -γ-globulin (BGG) formulation containing dextran was measured by size exclusion chromatography. The T1 of the BGG carbonyl carbon and dextran methin carbon in the formulation was determined by high resolution 13C NMR, and subsequently used to calculate the correlation time (τC) of each carbon. The spin-spin relaxation time (T2) of BGG and dextran protons was measured by pulsed NMR spectrometry, and the critical temperature of appearance of Lorentzian relaxation due to liquid BGG and dextran protons (Tmc) was determined. Results. The τC of dextran methin carbon in BGG-dextran formulations exhibited a linear temperature dependence according to the Adam-Gibbs-Vogel equation at lower temperatures, and a nonlinear temperature dependence described by the Vogel-Tamman-Fulcher equation at higher temperatures. The temperature at which molecular motion of dextran changed was consistent with the Tmc. The τC of BGG carbonyl carbon exhibited a similar temperature dependence to the τC of the dextran methin carbon and substantially decreased at temperatures above Tmc in the presence of dextran. The temperature dependence of BGG aggregation could be described by the Williams-Landel-Ferry equation even at temperatures 20°C lower than Tmc. Conclusions. High resolution 13C solid-state NMR indicated that the molecular motion of BGG was enhanced above Tmc in association with the increased global segmental motion of dextran molecules.

Molecular miscibility of phosphatidylcholine and phosphatidylethanolamine in binary mixed bilayers with acidic phospholipids studied by 2H- and 31P-NMR

The intermolecular interactions and microscopic miscibility of the lipid bilayers of single component and binary mixtures with high content of saturated fatty acids were investigated by 2H- and 31P-NMR for phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). Their glycerol backbones were selectively deuterated by biosynthesis and chemical synthesis. Deuterium quadrupole splittings and phosphorus chemical shift anisotropies provided the consistent information for the molecular miscibility of each phospholipids. PE was found to be completely miscible with PG and CL. Since deuterium quadrupole splittings and phosphorus chemical shift anisotropy are identical for two components in the mixed bilayer, the dynamic structure from the glycerol backbone to phosphate group should be uniform in the binary mixture of these phospholipids. In contrast to PE, PC was not fully miscible with PG and CL at molecular resolution. The dynamic structure from the glycerol backbone to phosphate group is different for two components in the binary mixed bilayers. In the case of the mixed bilayers of PC and PE, both phospholipids are microscopically immiscible with each other. Thus, while PE, PG and CL can adapt to a new situation to form a uniform dynamic structure in mixed bilayers, PC has no ability for adaptation. The molecular miscibility in lipid bilayers was shown to depend on the molecular species and the nature of the molecular interactions. The biological significance of this result was discussed.

Multidimensional solid-state nuclear magnetic resonance for determining the dihedral angle from the correlation of 13C–1H and 13C–13C dipolar interactions under magic-angle spinning conditions

Multidimensional solid-state nuclear magnetic resonance (NMR) under magic-angle spinning (MAS) conditions has been developed to determine the dihedral angle for a 1Hα–13Cα–13Cβ–1Hβ moiety in powdered states. The pulse sequence for this experiment includes 13C1H dipolar evolution periods for Cα and Cβ, which are correlated through a coherent 13Cα13Cβ dipolar mixing period. Theoretical analysis based on the symmetry of the spin system indicates that the dipolar correlation spectrum only due to the CαHα and CβHβ dipolar couplings is strongly dependent on the dihedral angle χ about the CαCβ bond axis, but two χ angles give the same spectrum in the χ range from 0° to about 140°, where χ=0° corresponds to the cis conformation. Inclusion of the CαCβ dipolar coupling together with the weak CαHβ and CβHα dipolar couplings, however, breaks the symmetry of the system with respect to χ in the range from 0° to 180°. These properties are confirmed by the spectra calculated for the pulse sequence as a function of χ and ...

The interactions of ferric and ferrous cytochrome c with cardiolipin in phospholipid membranes studied by solid-state 2H and 31P NMR

Abstract The interactions of ferric and ferrous cytochrome c with cardiolipin (CL) in lipid bilayers were investigated by solid-state 2 H and 31 P NMR. To examine the effect of the interaction on the glycerol backbone of CL, its glycerol moiety was specifically deuterated. 2 H NMR experiment showed that only ferricytochrome c interacts strongly with CL in the CL bilayers and binary mixed phosphatidylcholine(PC)/CL bilayers. This was consistent with the result of cytochrome c binding experiment. Ferricytochrome c binds to CL liposomes as much as two times of ferrocytochrome c . This suggests that the charge of the heme iron is also involved in the interaction. The change of the deuterium quadrupole splitting of CL on binding of cytochrome c was larger for the single component CL bilayer than for the PC/CL bilayer, suggesting that a CL domain rather than a single molecule is responsible for the strong interaction with cytochrome c .

Modulation of the specific interaction of cardiolipin with cytochrome c by zwitterionic phospholipids in binary mixed bilayers; a 2H and 31P NMR study

Abstract The interaction of cytochrome c with binary phospholipid mixtures was investigated by solid-state 2 H and 31 P NMR. To examine the effect of interaction on the glycerol backbones, the glycerol moieties of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) were specifically deuterated. On binding of cytochrome c to the binary mixed bilayers, no changes in the quadrupole splittings of each component were observed for PC/PG, PE/CL and PE/PG liposomes. In contrast, the splittings of CL decreased on binding of the protein to PC/CL liposomes, although those of PC did not change at all. This showed that cytochrome c specifically interacts with cardiolipin (CL) in phosphatidylcholine (PC)/CL bilayers and penetrates into the lipid bilayer to some extent so as to perturb the dynamic structure of the glycerol backbone. This is distinctly different from the mode of interaction of cytochrome c with other binary mixed bilayers. In 31 P NMR spectra, line broadening and a decrease of the chemical shift anisotropy were observed on binding of cytochrome c for all binary mixed bilayers examined. These changes were more significant for PC/CL bilayers. Furthermore, the line broadening is more significant for PC than for CL in PC/CL bilayers. Therefore, it can be concluded that as far as the polar head groups are concerned, not only CL but also PC are involved in the interaction with cytochrome c

Transient antibody-antigen interactions mediate the strain-specific recognition of a conserved malaria epitope

Transient interactions in which binding partners retain substantial conformational disorder play an essential role in regulating biological networks, challenging the expectation that specificity demands structurally defined and unambiguous molecular interactions. The monoclonal antibody 6D8 recognises a completely conserved continuous nine-residue epitope within the intrinsically disordered malaria antigen, MSP2, yet it has different affinities for the two allelic forms of this antigen. NMR chemical shift perturbations, relaxation rates and paramagnetic relaxation enhancements reveal the presence of transient interactions involving polymorphic residues immediately C-terminal to the structurally defined epitope. A combination of these experimental data with molecular dynamics simulations shows clearly that the polymorphic C-terminal extension engages in multiple transient interactions distributed across much of the accessible antibody surface. These interactions are determined more by topographical features of the antibody surface than by sequence-specific interactions. Thus, specificity arises as a consequence of subtle differences in what are highly dynamic and essentially non-specific interactions.Krishnarjuna et al. show that multiple transient interactions mediate monoclonal antibody recognition of an epitope within a disordered malaria antigen, MSP2. These results explain the antibody’s differential affinities for two allelic forms of the antigen.

Cold-Shock Expression System in E. coli for Protein NMR Studies

The cold-shock system using the pCold vector is one of the most effective Escherichia coli heterologous protein expression systems. It allows the improvement of the expression level of the protein of interest in a soluble fraction. In this chapter, we describe practical procedures for the overexpression of heterologous protein of interest by using the pCold vector or the single-protein production system. The latter is one of the most advanced pCold technologies for isotope labeling of the target protein and its NMR studies.

CHAPTER 3:13C–13C Distance Measurements by Polarization Transfer Matrix Analysis of 13C Spin Diffusion in a Uniformly 13C-Labeled Molecular Complex under Magic Angle Spinning

Spin diffusion of 13C polarization in NMR provides 13C−13C distances under magic angle spinning over a broad spectral width. However, there are difficulties in obtaining the distances accurately in uniformly 13C-labeled molecular complexes in solids. Effects of the weak long-range couplings are suppressed by strong short-range couplings. In addition, direct polarization transfer should be distinguished from relayed transfer. To address these issues, polarization-transfer rate matrix analysis has been applied to the 13C-driven spin diffusion in a uniformly 13C-labeled bacteriochlorophyll c assembly. The transfer rates due to direct dipolar couplings were derived by matrix analysis. Distances were obtained from the rates by perturbation theory for spin diffusion using zero-quantum lineshapes. This procedure gave distances up to 6 A with an accuracy of 25−50%. Correction of the distances from the zero-quantum lineshapes improved the accuracy by about 5−15%. These results show that rate matrix analysis is beneficial for distance analysis of molecular complexes for solid-state NMR. Also, the coefficient and anisotropy of 13C spin diffusion in solids are discussed quantitatively.

Microdomain formation in phosphatidylethanolamine bilayers detected by 2H-NMR.

In deuterium NMR spectra of phosphatidylethanolamine bilayers with an extremely high content of saturated fatty acids, each C1 deuteron of the glycerol backbone gave rise to a doublet [Yoshikawa et al., (1988) Biochim. Biophys. Acta 944, 321-328]. This suggests the presence of two backbone conformations, the exchange between which is slow on an NMR time-scale. The origin of the two conformations has been investigated in this work using saturated 1,2-diacyl-sn-glycero-3-phosphoethanolamine specifically deuterated in the glycerol backbone. The results showed that the two conformations originate from different domains, which have different fatty acid compositions. The differential scanning calorimetry of the bilayers suggested that the size of the domain is not large enough to show an independent phase transition. Thus, the formation of microdomains in the phosphatidylethanolamine bilayers has been concluded. Conformational difference in different domains was shown to be restricted to the C1 position of the glycerol backbone. The microdomains of phosphatidylethanolamine were retained even in the presence of other phospholipids.