Crystal structure, optical, magnetic, and photochemical properties of the complex pentakis(dimethyl sulfoxide)nitrosylchromium(2+) hexafluorophosphate

The nitrosyl complex [Cr(dmso)₅(NO)](PF₆)₂ (1) (dmso = dimethyl sulfoxide) has been prepared by the solvolysis of [Cr(NCCH₃)₅(NO)](PF₆)₂ in neat dmso. The optical absorption spectrum of 1 in dmso shows maxima at 734, 567, 450, 413, and 337 nm. Continuous photolysis of 1 with λ = 365-580 nm light in dmso solution results in a release of NO with quantum yield, Φ, in the range 0.034-0.108 mol Einstein⁻¹. Irradiation of a deoxygenated CH₃CN solution of [Cr(NCCH₃)₅(NO)](PF₆)₂ in the presence of excess of [Fe(S₂CNEt₂)₂] results in a transfer of NO to the iron centre as shown from the characteristic EPR spectrum of [Fe(S₂CNEt₂)₂(NO)] with A_iso(¹⁴N) = 12.2 × 10⁻⁴ cm⁻¹. The EPR parameters of 1 were determined: g_iso, g_‖ and g_⊥ : 1.96725, 1.91881(4) and 1.992763(2); A_iso(⁵³Cr), A_‖ (⁵³Cr) and A_⊥(⁵³Cr): 22.8 × 10⁻⁴, 39 × 10⁻⁴ and 15.8 × 10⁻⁴ cm⁻¹; A_iso(¹⁴N), A_‖ (¹⁴N) and A_⊥(¹⁴N): 5.9 × 10⁻⁴, 2 × 10⁻⁴ and 7.540(4) × 10⁻⁴ cm⁻¹.     

Optical and EPR spectra of the thionitrosyl complex [Cr(OH2)5(NS)]

The green thionitrosyl complex [Cr(OH₂)₅(NS)]²⁺ was isolated in solution by the hydrolysis of [Cr(NCCH₃)₅(NS)]²⁺. The optical absorption spectra of both compounds are dominated by a band with vibrational progression around 600 nm assigned as a {d_yz,zx, π^∗(NS)} → {π^∗(NS), d_yz,zx}^∗ transition. The optical data indicate that the NS ligand is a weaker π-acceptor than the NO ligand. The EPR parameters of [Cr(OH₂)₅(NS)]²⁺ were determined: g_iso, g_∥ and g_⊥: 1.96515, 1.92686(5) and 1.986860(8); A_iso(⁵³Cr), A_∥(⁵³Cr) and A_⊥(⁵³Cr): 25.3 × 10⁻⁴, 38 × 10⁻⁴ and 18.5 × 10⁻⁴ cm⁻¹; A_iso(¹⁴N), A_∥(¹⁴N) and A_⊥(¹⁴N): 6.5 × 10⁻⁴, 2.81 × 10⁻⁴ and 8.346(12) × 10⁻⁴ cm⁻¹.     

Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T1ρ NMR Relaxation

A membrane-bound form of Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles was used as a molecular probe to quantify for the viscosity of the lipid membrane interior by measuring the uniaxial rotational diffusion coefficient of the protein. Orientationally dependent ¹⁵N NMR relaxation times in the rotating frame, or T_1ρ, were determined by fitting individually the decay of the resolved NMR peaks corresponding to the transmembrane helix of Pf1 coat protein as a function of the spin-lock time incorporated into the 2D SAMPI4 pulse sequence. The T_1ρ relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sin⁴θ_B, where θ_B is the angle that the NH bond forms with respect to the axis of rotation. Importantly, the bond factors can be independently measured from the dipolar couplings in the separated local-field SAMPI4 spectra. From this dependence, the value of the diffusion coefficient D_|| = 8.0 × 10⁵ s^?1 was inferred from linear regression of the experimental T_1ρ data even without any spectroscopic assignment. Alternatively, a close value of D_|| = 7.7 × 10⁵ s^?1 was obtained by fitting the T_1ρ relaxation data for the assigned NMR peaks of the transmembrane domain of Pf1 to a wavelike pattern as a function of residue number. The method illustrates the use of single-helix transmembrane peptides as molecular probes to assess the dynamic parameters of biological membranes by NMR relaxation in oriented lipid bilayers.     

Amide Proton Spin-Lattice Relaxation in Polypeptides: A Field-Dependence Study of the Proton and Nitrogen Dipolar Interactions in Alumichrome

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the ¹H spinlattice relaxation times (T₁'s) are distinct in the Al³⁺-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of ¹H—¹H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T₁^-1 and Σr^-6 at the three magnetic fields. Considering the very sensitive r-dependence of T₁, the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T₁^-1 is proposed to mainly originate from the ¹H-¹⁴N dipolar interaction: this was supported by comparison with measurements on an ¹⁵N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {¹H}—¹H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the ¹H—¹H dipolar mechanism to cause NOE's. From the slope and intersect values of T₁^-1 vs. Σr^-6 linear plots, a number of independent estimates of τ_r, the rotational correlation time, were derived. These and the field-dependence of the T₁'s yield a best estimate <τ_r> ≈ 0.37 ns, in good agreement with 0.38 ns [unk] <τ_r> [unk] 0.41 ns, previously determined from ¹³C and ¹⁵N spin-lattice relaxation data.     

Copper(II) complexes as superoxide dismutase mimics: Synthesis, characterization, crystal structure and bioactivity of copper(II) complexes

Two new copper(II) complexes of the type [Cu(L)X₂), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elemental analyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivity measurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. The structures of these complexes determined by single crystal X-ray crystallography show a distorted square based pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN₂OX (X = Cl/Br) basal plane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligand and a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of these complexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g_|| > g_⊥ > 2.00) suggest that the unpaired electron on copper(II) has d_x²-y² character. Biological activities in terms of superoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been measured. The superoxide dismutase activity reveals that these two complexes catalyze the fast disproportionation of superoxide in DMSO solution.     

Lipid bilayer structure determined by the simultaneous analysis of neutron and X-ray scattering data

Quantitative structures were obtained for the fully hydrated fluid phases of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers by simultaneously analyzing x-ray and neutron scattering data. The neutron data for DOPC included two solvent contrasts, 50% and 100% D₂O. For DPPC, additional contrast data were obtained with deuterated analogs DPPC_d62, DPPC_d13, and DPPC_d9. For the analysis, we developed a model that is based on volume probability distributions and their spatial conservation. The model's design was guided and tested by a DOPC molecular dynamics simulation. The model consistently captures the salient features found in both electron and neutron scattering density profiles. A key result of the analysis is the molecular surface area, A. For DPPC at 50°C A = 63.0 Å², whereas for DOPC at 30°C A = 67.4 Å², with estimated uncertainties of 1 Å². Although A for DPPC agrees with a recently reported value obtained solely from the analysis of x-ray scattering data, A for DOPC is almost 10% smaller. This improved method for determining lipid areas helps to reconcile long-standing differences in the values of lipid areas obtained from stand-alone x-ray and neutron scattering experiments and poses new challenges for molecular dynamics simulations.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Homoleptic copper(II) and copper(I) complexes of 5,6-dihydro-5,6-epoxy-1,10-phenanthroline. Six-coordinate copper(I) in solution

Reaction of 5,6-dihydro-5,6-epoxy-1,10-phenanthroline (L) with Cu(ClO₄)₂·6H₂O in methanol in 3:1 M ratio at room temperature yields light green [CuL₃](ClO₄)₂·H₂O (1). The X-ray crystal structure of the hemi acetonitrile solvate [CuL₃](ClO₄)₂·0.5CH₃CN has been determined which shows Jahn-Teller distortion in the CuN₆ core present in the cation [CuL₃]²⁺. Complex 1 gives an axial EPR spectrum in acetonitrile-toluene glass with g_|| = 2.262 (A_|| = 169 × 10⁻⁴ cm⁻¹) and g_⊥ = 2.069. The Cu(II/I) potential in 1 in CH₂Cl₂ at a glassy carbon electrode is 0.32 V versus NHE. This potential does not change with the addition of extra L in the medium implicating generation of a six-coordinate copper(I) species [CuL₃]⁺ in solution. B3LYP/LanL2DZ calculations show that the six Cu-N bond distances in [CuL₃]⁺ are 2.33, 2.25, 2.32, 2.25, 2.28 and 2.25 Å while the ideal Cu(I)-N bond length in a symmetric Cu(I)N₆ moiety is estimated as 2.25 Å. Reaction of L with Cu(CH₃CN)₄ClO₄ in dehydrated methanol at room temperature even in 4:1 M proportion yields [CuL₂]ClO₄ (2). Its ¹H NMR spectrum indicates that the metal in [CuL₂]⁺ is tetrahedral. The Cu(II/I) potential in 2 is found to be 0.68 V versus NHE in CH₂Cl₂ at a glassy carbon electrode. In presence of excess L, 2 yields the cyclic voltammogram of 1. From ¹H NMR titration, the free energy of binding of L to [CuL₂]⁺ to produce [CuL₃]⁺ in CD₂Cl₂ at 298 K is estimated as −11.7 (±0.2) kJ mol⁻¹.     

Electron-electron distances in spin-labeled low-spin metmyoglobin variants by relaxation enhancement

Thirteen single-cysteine variants of myoglobin were prepared by overexpression of apoprotein, spin labeling, and reconstitution with hemin. This procedure resulted in a protein with fewer hemichrome impurities than was obtained by an overexpression of holo-protein followed by spin labeling. Coordination of cyanide to the met heme formed low-spin complexes. Iron-nitroxyl interspin distances in the range of 17-30 Å were determined by saturation recovery measurements of the enhancement of the nitroxyl spin lattice relaxation rates between ∼30-140 K, and by spin-echo measurements of the enhancement of spin-spin relaxation rates at 10-30 K. Interspin distances were also calculated, using the molecular modeling program Insight II (Accelrys, San Diego, CA). For most variants, distances determined from the temperature dependence of spin-echo intensities at a pulse spacing of 200 ns agree with distances measured by saturation recovery and calculated with Insight II within about an angstrom, which is within experimental uncertainties. Measurements of interspin distances via spin-spin relaxation enhancement have the advantages that maximum effects are observed for slower metal relaxation rates than are required for spin-lattice relaxation enhancement, and the impact diminishes as r⁻³ instead of r⁻⁶, as with spin-lattice relaxation enhancement, which permits measurements at longer distances.     

NMR studies on domain diffusion and alignment in modular GB1 repeats

Modular proteins contain individual domains that are often connected by flexible, unstructured linkers. Using a model system based on the GB1 domain, we constructed tandem repeat proteins and investigated the rotational diffusion and long-range angular ordering behavior of individual domains by measuring NMR relaxation parameters and residual dipolar couplings. Although they display almost identical protein-solvent interfaces, each domain exhibits distinct rotational diffusion and alignment properties. The diffusion tensor anisotropy of the N-terminal domain (NTD) is D_‖/D_⊥ = 1.5-1.6, similar to that of single-GB1 domains (D_‖/D_⊥ = 1.6-1.7), whereas the value for the C-terminal domain (CTD) is D_‖/D_⊥ = 2.0-2.2. In addition, the two domains have different rotational correlation times. These effects are observed for linkers of three to 24 residues, irrespective of linker length. The NTD and CTD also differ in their degree of magnetic alignment, even with a flexible linker of 18 residues, exhibiting D_a values of 7.7 Hz and 9.7 Hz, respectively. Our results suggest that diffusion differences and long-range influences may persist in modular protein systems, even for systems that have highly flexible linkers and exhibit no domain-domain or domain-linker interactions.     

A systematic molecular dynamics simulation study of temperature dependent bilayer structural properties

Although lipid force fields (FFs) used in molecular dynamics (MD) simulations have proved to be accurate, there has not been a systematic study on their accuracy over a range of temperatures. Motivated by the X-ray and neutron scattering measurements of common phosphatidylcholine (PC) bilayers (Ku?erka et al. BBA. 1808: 2761, 2011), the CHARMM36 (C36) FF accuracy is tested in this work with MD simulations of six common PC lipid bilayers over a wide range of temperatures. The calculated scattering form factors and deuterium order parameters from the C36 MD simulations agree well with the X-ray, neutron, and NMR experimental data. There is excellent agreement between MD simulations and experimental estimates for the surface area per lipid, bilayer thickness (D_B), hydrophobic thickness (D_C), and lipid volume (V_L). The only minor discrepancy between simulation and experiment is a measure of (D_B − D_HH) / 2 where D_HH is the distance between the maxima in the electron density profile along the bilayer normal. Additional MD simulations with pure water and heptane over a range of temperatures provide explanations of possible reasons causing the minor deviation. Overall, the C36 FF is accurate for use with liquid crystalline PC bilayers of varying chain types and over biologically relevant temperatures.     

Kinetics and mechanism of the reactions of ozone with superoxo, hydroperoxo, and hydrido complexes of rhodium(III)

Oxidation of the title complexes with ozone takes place by hydrogen atom, hydride, and electron transfer mechanisms. The reaction with (NH₃)₄(H₂O)RhH²⁺ is a two electron process, believed to involve hydride transfer with a rate constant k = (2.2 ± 0.2) × 10⁵ M⁻¹ s⁻¹ and an isotope effect k_H/k_D = 2. The oxidation of (NH₃)₄(H₂O)RhOOH²⁺ to (NH₃)₄(H₂O)RhOO²⁺ by an apparent hydrogen atom transfer is quantitative and fast, k = (6.9 ± 0.3) × 10³ M⁻¹ s⁻¹, and constitutes a useful route for the preparation of the superoxo complex. The latter is also oxidized by ozone, but more slowly, k = 480 ± 50 M⁻¹ s⁻¹.     

SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins

An immobilization procedure for protein on surface plasmon resonance sensor (SPR) chips is described. The target protein, cyclophilin D, is thereby genetically linked to a mutant of the human DNA repair protein O⁶-alkylguanine-DNA-alkyltransferase (hAGT). The procedure includes the immobilization of an alkylguanine derivative on the surface by amine coupling and contact of the surface with a solution of the fusion protein (TCypD-hAGT). TCypD-hAGT could be immobilized using buffer solutions of purified protein or cell extracts. High densities of covalently linked proteins were achieved by either procedure. Binding experiments performed with the ligand cyclosporin A indicate relative binding activities close to 100%. The K_D value (12 nM) and the kinetic rate constants k_on (3 × 10⁵ M⁻¹s⁻¹) and k_off (4 × 10⁻³s⁻¹) are given and compared to values determined for cyclophilin D linked to the surface by amide coupling chemistry. The K_D value is in excellent agreement with the K_D value determined in solution by fluorescence titration.     

Trinuclear-based coordination compounds of Mn(II) and Co(II) with 4-amino-3,5-dimethyl-1,2,4-triazole and azide and thiocyanate anions: Synthesis, structure and magnetic properties

A 2D layer complex 1 and a linear trinuclear complex 2 with mixed ligands have been synthesized and characterized by elemental analyses, IR and single-crystal X-ray diffraction. In 1, the Mn(II) ions are six-coordinated and lie in distorted octahedron coordination environments. Complex 1 is connected into a 2D layer structure based on a linear trinuclear Mn₃(admtrz)₄(N₃)₆ (admtrz = 4-amino-3,5-dimethyl-1,2,4-triazole) building unit with either (6,3) topology when Mn1 cations as three-connected nodes or (4,4) network when the coordination trinuclear units being regarded as four connected nodes. In 2, the Co(II) ions are in slightly distorted octahedron coordination geometries. The magnetic behaviors are investigated in the temperature range 1.8-300 K. The magnetic susceptibility measurements show that the Mn(II) ions of complex 1 are weakly antiferromagnetically coupled with g = 1.98(1), J1 = −6.31(5) cm⁻¹ and J2 = −1.88(1) cm⁻¹. There is dominant zero field splitting (ZFS) effects with g values, g_// = 2.38(2) and g_⊥ = 4.96(4), indicated a significant presence of the spin-orbit coupling and magnetization experiment reveals large, uniaxial zero-field splitting parameters of D = −29.55 cm⁻¹ for 2.     

Using pulse field gradient NMR diffusion measurements to define molecular size distributions in glycan preparations

Glycans comprise perhaps the largest biomass in nature, and more and more glycans are used in a number of applications, including those as pharmaceutical agents in the clinic. However, defining glycan molecular weight distributions during and after their preparation is not always straightforward. Here, we use pulse field gradient (PFG) ¹H NMR self-diffusion measurements to assess molecular weight distributions in various glycan preparations. Initially, we derived diffusion coefficients, D, on a series of dextrans with reported weight-average molecular weights from about 5 kDa to 150 kDa. For each dextran sample, we analyzed 15 diffusion decay curves, one from each of the 15 major ¹H resonance envelopes, to provide diffusion coefficients. By measuring D as a function of dextran concentration, we determined D at infinite dilution, D_inf, which allowed estimation of the hydrodynamic radius, R_h, using the Stokes-Einstein relationship. A plot of log D_inf versus log R_h was linear and provided a standard calibration curve from which R_h is estimated for other glycans. We then applied this methodology to investigate two other glycans, an α-(1→2)-l-rhamnosyl-α-(1→4)-d-galacturonosyl with quasi-randomly distributed, mostly terminal β(1→4)-linked galactose side-chains (GRG) and an α(1→6)-d-galacto-β(1→4)-d-mannan (Davanat), which is presently being tested against cancer in the clinic. Using the dextran-derived calibration curve, we find that average R_h values for GRG and Davanat are 76 ± 6 × 10⁻¹⁰ m and 56 ± 3 × 10⁻¹⁰ m, with GRG being more polydispersed than Davanat. Results from this study will be useful to investigators requiring knowledge of polysaccharide dispersity, needing to study polysaccharides under various solution conditions, or wanting to follow degradation of polysaccharides during production.     

Effect of substituent dtp to optical properties of heterobimetallic M/Ag/S nest-shaped clusters (M = Mo, W)

Clusters [MoS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (1), [WS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (2) and [WOS₃Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (3) were synthesized by the reaction of (NH₄)₂MoS₄/(NH₄)₂WS₄, (NH₄)₂WOS₃ with Ag[S₂P(OPrⁱ)₂]. Their structures have been characterized by X-ray diffraction. The clusters consist of a distorted tetrahedral MS₄ (or MOS₃) (M = Mo, W) with three Ag atoms and three sulfur atom bridges (Fig. 1), and resemble roughly that of cubane-like clusters. The nonlinear optical (NLO) properties were studied with an 8 ns pulsed laser at 532 nm. Its optical response to the incident light exhibits good optical absorptive and refractive effects, with α₂ = 1.56 × 10⁻¹⁰ m W⁻¹, n₂ = 3.87 × 10⁻¹⁷ m² W⁻¹ for cluster 1; α₂ = 1.33 × 10⁻¹⁰ m W⁻¹, n₂ = 6.52 × 10⁻¹⁷ m² W⁻¹for cluster 2; and α₂ = 2.54 × 10⁻¹⁰ m W⁻¹, n₂ = 4.07 × 10⁻¹⁷ m² W⁻¹ for cluster 3 for a 1.56 × 10⁻⁴ mol dm⁻³ CH₂Cl₂ solution.     

H and C NMR studies on the temperature-dependent water and protein dynamics in hydrated elastin,myoglobin and collagen

²H NMR spin-lattice relaxation and line-shape analyses are performed to study the temperature-dependent dynamics of water in the hydration shells of myoglobin, elastin, and collagen. The results show that the dynamical behaviors of the hydration waters are similar for these proteins when using comparable hydration levels of h = 0.25–0.43. Since water dynamics is characterized by strongly nonexponential correlation functions, we use a Cole–Cole spectral density for spin-lattice relaxation analysis, leading to correlation times, which are in nice agreement with results for the main dielectric relaxation process observed for various proteins in the literature. The temperature dependence can roughly be described by an Arrhenius law, with the possibility of a weak crossover in the vicinity of 220 K. Near ambient temperatures, the results substantially depend on the exact shape of the spectral density so that deviations from an Arrhenius behavior cannot be excluded in the high-temperature regime. However, for the studied proteins, the data give no evidence for the existence of a sharp fragile-to-strong transition reported for lysozyme at about 220 K. Line-shape analysis reveals that the mechanism for the rotational motion of hydration waters changes in the vicinity of 220 K. For myoglobin, we observe an isotropic motion at high temperatures and an anisotropic large-amplitude motion at low temperatures. Both mechanisms coexist in the vicinity of 220 K. ¹³C CP MAS spectra show that hydration results in enhanced elastin dynamics at ambient temperatures, where the enhancement varies among different amino acids. Upon cooling, the enhanced mobility decreases. Comparison of ²H and ¹³C NMR data reveals that the observed protein dynamics is slower than the water dynamics.     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques

Nisin, a peptide antibiotic, efficiently kills bacteria through a unique mechanism which includes inhibition of cell wall biosynthesis and pore formation in cytoplasmic membranes. Both mechanisms are based on interaction with the cell wall precursor lipid II which is simultaneously used as target and pore constituent. We combined two biosensor techniques to investigate the nisin activity with respect to membrane binding and pore formation in real time. Quartz crystal microbalance (QCM) allows the detection of nisin binding kinetics. The presence of 0.1 mol% lipid II strongly increased nisin binding affinity to DOPC (k_D 2.68 × 10^− 7 M vs. 1.03 × 10^− 6 M) by a higher association rate. Differences were less pronounced while using negatively charged DOPG membranes. However, lipid II does not influence the absolute amount of bound nisin. Cyclic voltammetry (CV) data confirmed that in presence of 0.1 mol% lipid II, nanomolar nisin concentrations were sufficient to form pores, while micromolar concentrations were necessary in absence of lipid II. Both techniques suggested unspecific destruction of pure DOPG membranes by micromolar nisin concentrations which were prevented by lipid II. This model membrane stabilization by lipid II was confirmed by atomic force microscopy. Combined CV and QCM are valuable to interpret the role of lipid II in nisin activity.