Cross correlation rates between Curie spin and dipole-dipole relaxation in paramagnetic proteins: the case of cerium substituted calbindin D9k

Cross correlation rates between Curie spin relaxation and H-N dipole-dipole coupling (^CS,DD _HM,HN) have been determined for a calcium binding protein, Calbindin D_9k, in which one of the two calcium ions is substituted with cerium(III). ^CS,DD _HM,HNvalues depend on both the metal-to-proton distances and the M-H-N angles and can be used as an additional constraint in order to refine the solution structure of paramagnetic metalloproteins. For this purpose, we have implemented a new module (CCR-DYANA) in a version of the program DYANA (PARAMAGNETIC-DYANA), which can be used together with other paramagnetism-based constraints such as pseudocontact shifts, residual dipolar couplings and hyperfine based Karplus relationships. This integrated structure calculation protocol has the advantage that different paramagnetic-based constraints are treated by the same algorithm in a way that the efficiency of each class of constraints can be analyzed and compared.     

Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel

A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring ¹⁵N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone ¹⁵N R ₁, R _1ρ, ¹⁵N–{¹H} NOE, and ¹⁵N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsA^TM, exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, τ_c = 38 ± 2.5 ns, at 50 °C and a diffusion anisotropy, , corresponding to an aspect ratio a/b ≥ 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S ² values in the 0.2–0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps–ns time scale for the 5-residue selectivity filter, which selects K⁺ ions to enter the channel. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . An erratum to this article can be found at     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.     

Electron paramagnetic resonance study of structural changes in the O photointermediate of bacteriorhodopsin

The structural changes of bacteriorhodopsin during its photochemical cycle, as revealed by crystal structures of trapped intermediates, have provided insights to the proton translocation mechanism. Because accumulation of the last photointermediate, O, appears to be hindered by lattice forces in the crystals, the only information about the structure of this state is from suggested analogies with the determined structures of the non-illuminated D85S mutant and wild-type bacteriorhodopsin at low pH. We used electron paramagnetic resonance spectroscopy of site-directed spin labels at the extracellular protein surface in membranes to test these models. Spin-spin dipolar interactions in the authentic O state compared to the non-illuminated state revealed that the distance between helices C and F increases by ca 4 Angstroms, there is no distance change between helices D and F, and the distance between helix D and helix B of the adjacent monomer increases. Further, the mobility changes of single labels indicate that helices E and F move outward from the proton channel at the center of the protein, and helix D tilts inward. The overall pattern of movements suggests that the model at acid pH is a better representation of the O state than D85S. However, the mobility analysis of spin-labels on the B-C interhelical loop indicates that the antiparallel beta-sheet maintains its ordered secondary structure in O, instead of the predicted disorder in the two structural models. During decay of the O state, the last step of the photocycle, a proton is transferred from Asp85 to proton release complex in the extracellular proton channel. The structural changes in O suggest the need of large conformational changes to drive the Arg82 side-chain back to its initial orientation towards Asp85, and to rearrange the numerous water molecules in this region in order to conduct the proton away from Asp85.     

Dielectric behavior of water in biological solutions: studies on myoglobin, human low-density lipoprotein, and polyvinylpyrrolidone

The dielectric behavior of the aqueous solutions of three widely differing macromolecules has been investigated: myoglobin, polyvinylpyrrolidone (PVP), and human serum low-density lipoprotein (LDL). It was not possible to interpret unambiguously the dielectric properties of the PVP solution in terms of water structure. The best interpretation of the dielectric data on the myoglobin and LDL solutions was that, in both cases, the macromolecule attracts a layer of water of hydration one or two water molecules in width. For LDL, this corresponds to a hydration factor of only 0.05 g/g, whereas for myoglobin the figure is nearer 0.6 g/g. With myoglobin, part of the water of hydration exhibits its dispersion at frequencies of a few GHz, and the rest disperses at lower frequencies, perhaps as low as 10-12 MHz. The approximate constancy of the width of the hydration shell for two molecules as dissimilar in size as LDL and myoglobin confirms that the proportion of water existing as water of hydration in a biological solution depends critically on the size of the macromolecules as well as on their concentration.     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.     

Similarities between intra- and intermolecular hydrogen bonds in RNA kissing complexes found by means of cross-correlated relaxation

The bond lengths and dynamics of intra- and intermolecular hydrogen bonds in an RNA kissing complex have been characterized by determining the NMR relaxation rates of various double- and triple-quantum coherences that involve an imino proton and two neighboring nitrogen-15 nuclei belonging to opposite bases. New experiments allow one to determine the chemical shift anisotropy of the imino protons. The bond lengths derived from dipolar relaxation and the lack of modulations of the nitrogen chemical shifts indicate that the intermolecular hydrogen bonds which hold the kissing complex together are very similar to the intramolecular hydrogen bonds in the double-stranded stem of the RNA.     

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions: The influence of in situ field gradients

The ¹H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T₂ values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with τ = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25–1.98 G/cm and 0.89–2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.     

Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X-and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V_i-bound biochemical state of myosin, which presumably mimics the S1.ADP.P_i state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V_i biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.     

Hatching fraction and timing of resting stage production in seasonal environments: effects of density dependence and uncertain season length

Many organisms survive unfavourable seasons as resting stages, some of which hatch each favourable season. Hatching fraction and timing of resting stage production are important life history variables. We model life cycles of freshwater invertebrates in temporary pools, with various combinations of uncertain season length and density‐dependent fecundity. In deterministic density‐independent conditions, resting stage production begins suddenly. With uncertain season length and density independence, resting stage production begins earlier and gradually. A high energetic cost of resting stages favours later resting stage production and a lower hatching fraction. Deterministic environments with density dependence allow sets of coexisting strategies, dominated by pairs, each switching suddenly to resting stage production on a different date, usually earlier than without density dependence. Uncertain season length and density dependence allow a single evolutionarily stable strategy, around which we observe many mixed strategies with negatively associated yield (resting stages per initial active stage) and optimal hatching fraction.     

Physiological and psychological effects of walking on young males in urban parks in winter

Background

Interaction with nature has a relaxing effect on humans. Increasing attention has been focused on the therapeutic effects of urban green space; however, there is a lack of evidence-based field research. This study provided scientific evidence supporting the physiological and psychological effects of walking on young males in urban parks in winter.

Findings

Subjects (13 males aged 22.5 ± 3.1 years) were instructed to walk predetermined 15-minute courses in an urban park (test) and in the city area (control). Heart rate and heart rate variability (HRV) were measured to assess physiological responses. The semantic differential (SD) method, Profile of Mood States (POMS), and State-Trait Anxiety Inventory (STAI) were used to determine psychological responses.Heart rate was significantly lower and the natural logarithm of the high frequency component of HRV was significantly higher when walking through the urban park than through the city area. The results of three questionnaires indicated that walking in the urban park improved mood and decreased negative feelings and anxiety.

Conclusions

Physiological and psychological data from this field experiment provide important scientific evidence regarding the health benefits of walking in an urban park. The results support the premise that walking in an urban park has relaxing effects even in winter.     

The effects of a weak permanent magnetic field on the lipid composition and content in the onion leaves of various ages

We have studied the effects of a weak permanent magnetic field (PMF) with strength of 403 A/m on the composition and content of polar and neutral lipids and the composition of their fatty acids (FAs). The lipids were isolated from the third, fourth, and fifth leaves of onion (Allium cepa L., cv. Arzamasskii) plants, and their composition was determined using TLC and GLC techniques. Plants growth under the conditions of a natural geomagnetic field served as a control. Most intense changes in the lipid content induced by PMF were observed in the fourth onion leaf. The content of total lipids and that of polar lipids (glyco-and phospholipids) changed, whereas the content of neutral lipids either decreased or remained unchanged. The phospholipid/sterol ratio increased, causing an increase in the fluidity of the membrane lipid bilayer. PMF induced an increase in the concentration of linolenic acid and the relative content of total unsaturated FAs. The effects of PMF on the content and composition of lipids in the third and fifth onion leaves were less pronounced, demonstrating differences between the leaves of various ages in their sensitivity to the effects of magnetic field. It is concluded that changes in the weak PMF within the limits of changes in the strength of geomagnetic field in the course of evolution can affect biochemical and physiological processes of plants.     

The constancy of the G matrix through species divergence and the effects of quantitative genetic constraints on phenotypic evolution: a case study in crickets

Long-term phenotypic evolution can be modeled using the response-to-selection equation of quantitative genetics, which incorporates information about genetic constraints (the G matrix). However, little is known about the evolution of G and about its long-term importance in constraining phenotypic evolution. We first investigated the degree of conservation of the G matrix across three species of crickets and qualitatively compared the pattern of variation of G to the phylogeny of the group. Second, we investigated the effect of G on phenotypic evolution by comparing the direction of greatest quantitative genetic variation within species (g(max)) to the direction of phenotypic divergence between species (Delta(z)). Each species, Gryllus veletis, G. firmus, and G. pennsylvanicus, was reared in the laboratory using a full-sib breeding design to extract quantitative genetic information. Five morphological traits related to size were measured. G matrices were compared using three statistical approaches: the T method, the Flury hierarchy, and the MANOVA method. Results revealed that the differences between matrices were small and mostly caused by differences in the magnitude of the genetic variation, not by differences in principal component structure. This suggested that the G matrix structure of this group of species was preserved, despite significant phenotypic divergence across species. The small observed differences in G matrices across species were qualitatively consistent with genetic distances, whereas ecological information did not provide a good prediction of G matrix variation. The comparison of g(max) and Delta(z) revealed that the angle between these two vectors was small in two of three species comparisons, whereas the larger angle corresponding to the third species comparison was caused in large part by one of the five traits. This suggests that multivariate phenotypic divergence occurred mostly in a direction predicted by the direction of greatest genetic variation, although it was not possible to demonstrate the causal relationship from G to Delta(z). Overall, this study provided some support for the validity of the predictive power of quantitative genetics over evolutionary time scales.     

Characterization of the sodium binding sites in microcrystalline ATP by Na-solid-state NMR and ab initio calculations

Stepwise conformational transition of disodium adenosine 5′-triphosphate (Na₂ATP) crystals as a function of relative humidity (r.H.), was examined by means of high resolution ²³Na, ³¹P solid state NMR spectroscopy and quantum mechanics calculations. ²³Na-MQMAS solid state NMR spectra of hydrated disodium salts of adenosine 5,5′-triphosphate were measured to characterize the individual sodium sites. Quadrupolar coupling constants (C_Q), and the asymmetry parameters of the electric field gradient tensors (η) of sodium-23 determined by quantum mechanical calculations (gaussian03) using larger clusters than in previous studies were compared to the experimental ²³Na-MQMAS spectra.     

A Tris-induced change in the midpoint potential of Z,the donor to photosystem II,as determined by the kinetics of the back reaction

A new method of measuring the rate of the back reaction from the state Z⁺ P680 Q_A^? in Tris-washed chloroplasts is described. By using ratios of back reaction rates we demonstrate a Tris-induced change in the equilibrium between Z and P680 and attribute this change to an alteration of the midpoint potential of Z by Tris treatment. We also demonstrate that the previously observed inhibition of the back reaction by ADRY reagents can be localized at Z and understood in terms of electron donation to Z⁺ by ADRY reagents.     

Solid-state NMR approaches to measure topological equilibria and dynamics of membrane polypeptides

Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the ¹⁵N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of ¹⁵N and ²H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs.     

H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin

The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN⁻ have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors. Ligand binding in heme-containing proteins is determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. Since both the RR Fe–OH⁻ and Fe–CN⁻ frequencies are very sensitive to the distal environment, detailed information on structural variations has been obtained. The hydroxyl ligand binds only the WT protein giving rise to two different conformers. In form 1 the anion is stabilized by H-bonds with TrpG8, TyrCD1 and a water molecule, in turn H-bonded to TyrB10. In form 2, H-bonding with TyrCD1 is mediated by a water molecule. Unlike the OH⁻ ligand, CN⁻ binds both WT and the triple mutant giving rise to two forms with similar spectroscopic characteristics. The overall results clearly indicate that H-bonding interactions both with distal residues and water molecules are important structural determinants in the active site of Tf-trHb. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Investigating the interaction of Grammostola rosea venom peptides and model lipid bilayers with solid-state NMR and electron microscopy techniques

Spider venom contains a number of small peptides that can control the gating properties of a wide range of ion channels with high affinity and specificity. These ion channels are responsible for coordination and control of many bodily functions such as transducing signals into sensory functions, smooth muscle contractions as well as serving as sensors in volume regulation. Hence, these peptides have been the topic of many research efforts in hopes that they can be used as biomedical therapeutics. Several peptides are known to control the gating properties of ion channels by involving the lipid membrane. GsMTx4, originally isolated from the Chilean Rose tarantula (Grammostola rosea), is known to selectively inhibit mechanosensitive ion channels by partitioning into the lipid bilayer. To further understand this indirect gating mechanism, we investigated the interactions between native GsAF2, VsTx1 and a synthetic form of GsMTx4 with model DMPC lipid bilayers using ³¹P solid-state NMR, ¹³C CP-MAS NMR, NS-TEM and cryo-TEM. The results reveal that these inhibitor cystine knot peptides perforate the DMPC lipid vesicles similarly with some subtle differences and ultimately create small spherical vesicles and anisotropic cylindrical and discoidal vesicles at concentrations near 1.0–1.5?mol% peptide. The anisotropic components align with their long axes along the NMR static B₀ magnetic field, a property that should be useful in future NMR structural investigations of these systems. These findings move us forward in our understanding of how these peptides bind and interact with the lipid bilayer.     

Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency

The fluorescence emission spectrum of Synechocystis sp. PPC6803 cells, at room temperature, displays: i) significant bandshape variations when collected under open (F₀) and closed (F_M) Photosystem II reaction centre conditions; ii) a marked dependence on the excitation wavelength both under F₀ and F_M conditions, due to the enhancement of phycobilisomes (PBS) emission upon their direct excitation. As a consequence: iii) the ratio of the variable and maximal fluorescence (F_V/F_M), that is a commonly employed indicator of the maximal photochemical quantum efficiency of PSII (Φ_{pc, PSII}), displays a significant dependency on both the excitation and the emission (detection) wavelength; iv) the F_V/F_M excitation/emission wavelength dependency is due, primarily, to the overlap of PSII emission with that of supercomplexes showing negligible changes in quantum yield upon trap closure, i.e. PSI and a PBS fraction which is incapable to transfer the excitation energy efficiently to core complexes. v) The contribution to the cellular emission and the relative absorption-cross section of PSII, PSI and uncoupled PBS are extracted using a spectral decomposition strategy. It is concluded that vi) Φ_{pc, PSII} is generally underestimated from the F_V/F_M measurements in this organism and, the degree of the estimation bias, which can exceed 50%, depends on the measurement conditions. Spectral modelling based on the decomposed emission/cross-section profiles were extended to other processes typically monitored from steady-state fluorescence measurements, in the presence of an actinic illumination, in particular non-photochemical quenching. It is suggested that vii) the quenching extent is generally underestimated in analogy to F_V/F_M but that viii) the location of quenching sites can be discriminated based on the combined excitation/emission spectral analysis.