Side-chain conformers to allow conversion from normal to isoaspartate in age-related proteins and peptides.

Dß (or D-iso)- and Lß- (or iso)- aspartyl (Asp) residues are accumulated in aged lens crystallins and amyloid beta (Aß) proteins, respectively, as a result of spontaneous, nonenzymatic isomerization of normal Lα-Asp. To explore why such uncommon Asp isomers are accumulated, the stability of Lα-, Lß-, and Dß-Asp was compared in view of the staggered side-chain conformers. By using cylindrin (KVKVLGD7VIEV) from αB-crystallin and Aß17-25 (L17VFF20AED23)VG25) containing Asp isomers, the vicinal spin-spin coupling constants of Asp Hα-Hß1 and Hα-Hß2 were quantified by high-resolution solution 1H NMR. It was found that the trans conformer was extremely preferred in Dß-Asp7 side-chain of cylindrin. In Aß17-25, the side chain of Lß-Asp23 was likely to adopt trans conformer, while gauche conformers were rather rich in Lα-Asp23. In gauche conformers, the close distance between Asp carboxylate carbon (CCOO-) and backbone nitrogen (N) next to Asp is advantageous to the intramolecular cyclization to form succinimide intermediate, followed by the conversion from α- to ß-Asp. The cyclization is limited in the trans conformer because of the long distance between CCOO- and N, to keep Dß- or Lß-Asp stable. This would be the reason for the site specificity of Asp isomerization in proteins. The higher population of trans conformer in Asp side chain, the less isomerization of Asp as shown as Asp76 in αA-crystallin. The stability and less reactivity of normal Asp and its isomers are the potential factors to determine whether or not the abnormal accumulation is permitted in aged crystallins and Aß.

Solution NMR to Quantify Mobility in Membranes: Diffusion, Protrusion, and Drug Transport Processes.

Lipid bilayer membranes are soft, fluid, and dynamic architecture where molecules are constantly moving and thermally fluctuating under physiological conditions. In this review, a strategy to quantify molecular dynamics in membranes is introduced by utilizing solution-state NMR spectroscopy as a versatile, noninvasive technique. The dynamics involves lateral diffusion and protrusion motion, in parallel and vertical direction to the membrane surface. Dynamical behavior of small-sized drugs, chemicals, and peptides is also reviewed in relation to the lipid movements in membranes, on the basis of recent multinuclear NMR in combination with the pulsed field gradient technique. Finally, in-cell NMR method is introduced as a promising technique to capture drug transport processes in real time, to shed light on mechanisms of deliveries to living cells without perturbation of the system.

Evaluation of the diameter of the distal radial artery at the anatomical snuff box using ultrasound in Japanese patients.

Catheter angioplasty or angiography via the distal access point of the radial artery (dRA), located at the anatomical snuff box, is a less invasive strategy for coronary intervention attracting considerable attention. Determining the diameter of the dRA is necessary to minimize the risk of artery occlusion and safely perform catheter intervention. This was a retrospective observational study including patients who underwent coronary angiography or coronary intervention at Aomori Kyoritsu Hospital, Aomori, Japan, between February 2018 and August 2018. The diameter of the dRA and the conventional access point of the radial artery (cRA) at the wrist of the patients were measured using ultrasound prior to angiography or interventional procedure. A total of 120 patients were analyzed. In male patients, the diameters of the cRA and dRA were 2.62 ± 0.60 mm and 2.04 ± 0.43 mm, respectively. In females, these diameters were 2.44 ± 0.51 mm and 1.96 ± 0.44 mm, respectively. Overall, the dRA was statistically significantly smaller than the cRA. However, variations were observed, with eight patients (6.7%) having a larger dRA than cRA. The diameter of the dRA indicated only that of the cRA. A multivariate analysis did not reveal factors associated with vessel diameter. The size and anatomy of the dRA varied considerably. Thus, it is difficult to predict the actual diameter of the artery. Customized selection of the size of the sheath and site of intervention is essential for each patient to safely perform ultrasound examination prior to cannulation.

Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.

Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell surface have recently been suggested to involve in not only endocytic cellular uptake but also non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides (CPPs). However, in-situ comprehensive observation and the quantitative analysis of the direct membrane translocation processes are challenging, and the mechanism therefore remains still unresolved. In this work, real-time in-cell NMR spectroscopy was applied to investigate the direct membrane translocation of octaarginine (R8) into living cells. By introducing 4-trifluoromethyl-l-phenylalanine to the N terminus of R8, the non-endocytic membrane translocation of 19F-labeled R8 (19F-R8) into a human myeloid leukemia cell line was observed at 4 °C with a time resolution in the order of minutes. 19F NMR successfully detected real-time R8 translocation: the binding to anionic GAGs at the cell surface, followed by the penetration into the cell membrane, and the entry into cytosol across the membrane. The NMR concentration analysis enabled quantification of how much of R8 was staying in the respective translocation processes with time in situ. Taken together, our in-cell NMR results provide the physicochemical rationale for spontaneous penetration of CPPs in cell membranes.

Kinetics of the competitive reactions of isomerization and peptide bond cleavage at l-α- and d-ß-aspartyl residues in an αA-crystallin fragment.

d-ß-aspartyl (Asp) residue has been found in a living body such as aged lens crystallin, although l-α-amino acids are constituents in natural proteins. Isomerization from l-α- to d-ß-Asp probably modulates structures to affect biochemical reactions. At Asp residue, isomerization and peptide bond cleavage compete with each other. To gain insight into how fast each reaction proceeds, the analysis requires the consideration of both pathways simultaneously and independently. No information has been provided, however, about these competitive processes because each reaction has been studied separately. The contribution of Asp isomers to the respective pathways has still been veiled. In this work, the two competitive reactions, isomerization and spontaneous peptide bond cleavage at Asp residue, were simultaneously observed and compared in an αA-crystallin fragment, S51 LFRTVLD58 SG60 containing l-α- and d-ß-Asp58 isomers. The kinetics showed that the formation of l- and d-succinimide (Suc) intermediate, as a first step of isomerization, was comparable at l-α- and d-ß-Asp. Although l-Suc was converted to l-ß-Asp, d-Suc was liable to return to the original d-ß-Asp, the reverse reaction marked enough to consider d-ß-Asp as apparently stable. d-ß-Asp was also resistant to the peptide bond cleavage. Such apparent less reactivity is probably the reason for gradual and abnormal accumulation of d-ß-Asp in a living body under physiological conditions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

D-ß-aspartyl residue exhibiting uncommon high resistance to spontaneous peptide bond cleavage.

Although L-amino acids were selected as main constituents of peptides and proteins during chemical evolution, D-aspartyl (Asp) residue is found in a variety of living tissues. In particular, D-ß-Asp is thought to be stable than any other Asp isomers, and this could be a reason for gradual accumulation in abnormal proteins and peptides to modify their structures and functions. It is predicted that D-ß-Asp shows high resistance to biomolecular reactions. For instance, less reactivity of D-ß-Asp is expected to bond cleavage, although such information has not been provided yet. In this work, the spontaneous peptide bond cleavage was compared between Asp isomers, by applying real-time solution-state NMR to eye lens αΑ-crystallin 51-60 fragment, S(51)LFRTVLD(58)SG(60) and αΒ-crystallin 61-67 analog, F(61)D(62)TGLSG(67) consisting of L-α- and D-ß-Asp 58 and 62, respectively. Kinetic analysis showed how tough the uncommon D-ß-Asp residue was against the peptide bond cleavage as compared to natural L-α-Asp. Differences in pKa and conformation between L-α- and D-ß-Asp side chains were plausible factors to determine reactivity of Asp isomers. The present study, for the first time, provides a rationale to explain less reactivity of D-ß-Asp to allow abnormal accumulation.

Staggered side-chain conformers of aspartyl residues prerequisite to transformation from L-α- to D-ß-aspartate 58 in human-lens αA-crystallin fragment.

D-ß-aspartyl (Asp) residues are found in aged human-lens αA-crystallin. To explore why the uncommon D-ß-Asp is accumulated, the stability of L-α-, D-α-, and D-ß-Asp residues is compared in view of the staggered side-chain conformers. By using αA-crystallin fragment, T(55)VLD(58)SGISEVR(65), composed of Asp58 isomers, the vicinal spin-spin coupling constants of Asp58 Hα-Hß1 and Hα-Hß2 are quantified by high-resolution solution NMR. The trans conformer is most preferred in the D-ß-Asp side-chain, whereas gauche+ and gauche- are abundant in L-α- and D-α-Asp. The close distance between Asp58 carboxylate carbon (CCOO-) and Ser59 nitrogen (N) in gauche+ and gauche- is advantageous to the intramolecular cyclization to form succinimide intermediate, followed by the transformation from α- to ß-Asp. The cyclization is not allowed in the trans conformer because of the long distance between CCOO- and N, to keep D-ß-Asp stable. The change from gauche to trans conformer in D-ß-Asp is exothermic and enthalpy-driven.

Uptake of sevoflurane limited by the presence of cholesterol in the lipid bilayer membrane: a multinuclear nuclear magnetic resonance study.

The effect of cholesterol on the uptake of a fluorinated general anesthetic, sevoflurane (SF, fluoromethyl 2,2,2-trifluoro-1-[trifluoromethyl]ethyl ether) was studied by multinuclear, high-resolution nuclear magnetic resonance (NMR) spectroscopy in combination with a pulsed-field gradient technique. Using large unilamellar vesicles of egg phosphatidylcholine/egg phosphatidylglycerol/cholesterol as model fluid cell membranes, the (19)F and (1)H NMR chemical shifts, longitudinal relaxation times (T1), and diffusion coefficients (D(eff)) were systematically analyzed to quantify the modulation of SF uptake to the lipid membrane by cholesterol. All NMR parameters (chemical shift, T1, and D(eff)) showed that SF uptake is limited by the presence of cholesterol in the membrane. It was found that SF uptake at 40 mol% cholesterol is limited to 50%-60% of the partitioning fraction in the absence of cholesterol in the membrane. This finding is attributed to the loss of motional freedom in the rigid membrane environment, as demonstrated by the gradual slowdown of lipid mobility D(eff) with increase in cholesterol concentration from 0 mol% to 40 mol%.

Lateral diffusion of lipids separated from rotational and translational diffusion of a fluid large unilamellar vesicle.

A new method to separate lateral diffusion of lipids in spherical large unilamellar vesicles from the rotational and the translational diffusion of the vesicle as a whole is proposed. The lateral diffusion coefficient DL is obtained as a time-dependent part of the observed diffusion coefficient in vesicles of 800-nm diameters, by systematically changing the diffusion time interval of the high-field-gradient NMR measurement. Although the lipid is in a confined space, the DL of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is (1.5±0.6)×10(-11) m(2) s(-1) in the fluid state at 45°C, more than one order of magnitude faster than the rotational and the translational diffusion coefficients of the vesicle by the hydrodynamic continuum model. The method provides a potential for quantifying the lateral diffusion of lipids and proteins in fluid bilayer vesicles as model cell membranes in a natural manner.

Binding of hydrophobic fluorinated bisphenol A to large unilamellar vesicles of egg phosphatidylcholine.

The kinetics of membrane binding and dissociation of fluorinated bisphenol A (FBPA, (CF(3))(2)C(C(6)H(4)OH)(2)) is quantified by 1D (19)F NMR spectra in situ. Although the bound and free components are in fast exchange, the rate constants and bound fraction is nonetheless determined from an analysis of the spectra. The analysis relies on the expression of 1D NMR signal intensity by a set of Bloch equations with exchange terms. The time span of the binding and dissociation of hydrophobic FBPA to large unilamellar vesicles of egg phosphatidylcholine (EPC) is 10(-3) to 10(-2) s. The rates of FBPA binding and dissociation are kept constant per EPC molecule even at different concentrations of the vesicle. The free energy of FBPA transfer is -20 ± 2 kJ/mol at 303 K. The process is entropy-driven. The efficiency of FBPA transfer is enhanced by a factor of 7 × 10(4), as compared with the hydrophilic 5-fluorouracil.

Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient 1H NMR in situ.

The kinetics of binding, the diffusivity, and the binding amount of a neuropeptide, leucine-enkephalin (L-Enk) to lipid bilayer membranes are quantified by pulsed-field-gradient (PFG) 1H NMR in situ. The peptide signal is analyzed by the solution of the Bloch equation with exchange terms in the presence of large unilamellar vesicles (LUVs) as confined, but fluid model cell membranes. Even in the case that the membrane-bound and the free states of L-Enk cannot be distinguished in the one-dimensional NMR spectrum, the PFG technique unveils the bound component of L-Enk after the preferential decay of the free component at the high field gradient. In 100-nm diameter LUVs consisting of egg phosphatidylcholine, the rate constants of the peptide binding and dissociation are 0.040 and 0.40 s-1 at 303 K. This means that the lifetime of the peptide binding is of the order from second to ten-second. The diffusivity of the bound L-Enk is 5×10-12m2/s, almost 60 times as restricted as the movement of free L-Enk at 303K. One-tenth of 5mM L-Enk is bound to 40mM LUV. The binding free energy is calculated to be -2.9 kJ/mol, the magnitude close to the thermal fluctuation, 2.5 kJ/mol. The result demonstrates the potential of PFG 1H NMR to quantify molecular dynamics of the peptide binding to membranes.

Drug binding and mobility relating to the thermal fluctuation in fluid lipid membranes.

Drug binding and mobility in fluid lipid bilayer membranes are quantified in situ by using the multinuclear solution NMR combined with the pulsed-field-gradient technique. One-dimensional and pulsed-field-gradient (19)F and (1)H NMR signals of an anticancer drug, 5-fluorouracil (5FU) are analyzed at 283-313 K in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes. The simultaneous observation of the membrane-bound and free 5FU signals enables to quantify in what amount of 5FU is bound to the membrane and how fast 5FU is moving within the membrane in relation to the thermal fluctuation of the soft, fluid environment. It is shown that the mobility of membrane-bound 5FU is slowed down by almost two orders of magnitude and similar to the lipid movement in the membrane, the movement closely related to the intramembrane fluidity. The mobility of 5FU and EPC is, however, not similar at 313 K; the 5FU movement is enhanced in the membrane as a result of the loose binding of 5FU in the lipid matrices. The membrane-bound fraction of 5FU is approximately 0.1 and almost unaltered over the temperature range examined. It is also independent of the 5FU concentration from 2 to 30 mM with respect to the 40-50 mM LUV. The free energy of the 5FU binding is estimated at -4 to -2 kJ/mol, the magnitude always close to the thermal fluctuation, 2.4-2.6 kJ/mol.

Cholesterol location and orientation in aqueous suspension of large unilamellar vesicles of phospholipid revealed by intermolecular nuclear overhauser effect.

The locational and orientational structure and the dynamics of cholesterol in the bilayer membrane were studied by using the solution-state NMR. The intermolecular nuclear Overhauser effect (NOE) was analyzed for large unilamellar vesicles (100 nm in diameter) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol at cholesterol concentrations of 9-33 mol %. The DMPC headgroups show (1)H-{(1)H}-NOEs with the methyl groups at the hydrophobic terminals of both cholesterol and DMPC, illustrating the significant fluctuation of the bilayer membrane in the vertical (bilayer normal) direction. Cholesterol was found to keep the hydroxyl (OH) group toward the outer water pool on the basis of the following observations: (1) the cross correlation between the DMPC headgroup and the cholesterol terminal methyl group is weaker than those between the DMPC headgroups and (2) the methyl group at the hydrophobic terminal of cholesterol shows strong correlation with the terminal group of the DMPC chain portion. The OH group plays a crucial role in orienting cholesterol with its OH group outward, since cholestane, which has a molecular structure similar to that of cholesterol except for the absence of the OH group, was found to have no orientational preference in the bilayer membrane. The dynamic slowdown at high cholesterol concentrations is demonstrated on the basis of the correlation times for NOE as well as the broadening of the proton linewidths.

Dynamic and 2D NMR studies on hydrogen-bonding aggregates of cholesterol in low-polarity organic solvents.

Self-diffusion coefficients (D) are measured for normal (nondeuterated) and deuterated cholesterol-d(6) (C26 and C27 methyl groups deuterated) in 1-octanol, chloroform, and cyclohexane at concentrations of 1-700 mM by varying the impurity water concentration (>2 mM) and temperature (30-50 degrees C). The pulsed field gradient spin-echo (PGSE) (1)H and (2)H NMR were used, respectively, at 600 and 92 MHz. At 30 degrees C, the hydrodynamic radius (R) obtained at 20 mM from the D value and solvent viscosity is 5.09, 7.07, and 6.17 A, respectively, in 1-octanol, chloroform, and cyclohexane when the impurity water is negligible. The R value in 1-octanol is the smallest and comparable with the average length of the molecular axes for the cholesterol molecule. In 1-octanol, R is invariant against the concentration variation, whereas in chloroform, R is larger and increases almost linearly with cholesterol concentration. At the highest concentration, 700 mM, the R in chloroform is 13.5 and 16.7 A, respectively, when the impurity water is at negligible and saturated concentrations. The R value larger than that in hydrogen-bonding 1-octanol indicates that cholesterol forms an aggregate through hydrogen bonding. The aggregate structure is confirmed by comparing NOESY spectra in chloroform and 1-octanol. The NOESY analysis reveals the presence of one extra cross peak (C4-C19) in chloroform compared to 1-octanol. Because the carbon atoms related to the cross peak are close to the hydroxyl group (C3-OH), cholesterol molecules are considered to be not piled but are found to be OH-centered in the aggregate. This is supported also by larger rotational hydrodynamic radii measured on cholesterol deuterated at positions C2, C3, C4, and C6. This shows that the aggregate formation is driven by the hydrogen-bonding between cholesterol molecules.

NMR study on the binding of neuropeptide achatin-I to phospholipid bilayer: the equilibrium, location, and peptide conformation.

Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance (13)C and high-resolution (of 0.01 Hz order) (1)H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal NH(3)(+) group in achatin-I; the de-ionization of NH(3)(+) decreases the bound fraction of the peptide from approximately 15% to nearly none. The electrostatic attraction between the N-terminal positive NH(3)(+) group and the negative PO(4)(-) group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the (13)C chemical shifts (delta) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Delta delta of the (13)C resonance for almost all the carbon sites in achatin-I. The changes Delta delta for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic CO(2)(-) groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic d-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.

Limited slowdown of endocrine-disruptor diffusion in confined fluid lipid membranes.

Self-diffusion rates of lipids and trapped bisphenol A (BPA) are determined in various sizes of confined but fluid membranes by high-field-gradient NMR at 600 MHz. Micelles and vesicles of 3- to 400-nm diameters are used as model membranes to get an insight into the molecular diffusion in such soft environments. The slowdown of BPA and lipid motions is leveled off in 100- and 400-nm vesicles, although the hydrodynamic continuum model gives the aggregate motion slowed inversely to the aggregate size. Instead, the limited motion is related to the intra-membrane fluidity.