Relating side-chain mobility in proteins to rotameric transitions: Insights from molecular dynamics simulations and NMR

The dynamic aspect of proteins is fundamental to understanding protein stability and function. One of the goals of NMR studies of side-chain dynamics in proteins is to relate spin relaxation rates to discrete conformational states and the timescales of interconversion between those states. Reported here is a physical analysis of side-chain dynamics that occur on a timescale commensurate with monitoring by ²H spin relaxation within methyl groups. Motivated by observations made from tens-of-nanoseconds long MD simulations on the small protein eglin c in explicit solvent, we propose a simple molecular mechanics-based model for the motions of side-chain methyl groups. By using a Boltzmann distribution within rotamers, and by considering the transitions between different rotamer states, the model semi-quantitatively correlates the population of rotamer states with ‘model-free’ order parameters typically fitted from NMR relaxation experiments. Two easy-to-use, analytical expressions are given for converting S²_axis’ values (order parameter for C–CH₃ bond) into side-chain rotamer populations. These predict that S²_axis’ values below 0.8 result from population of more than one rotameric state. The relations are shown to predict rotameric sampling with reasonable accuracy on the ps–ns timescale for eglin c and are validated for longer timescales on ubiquitin, for which side-chain residual dipolar coupling (RDC) data have been collected.     

Patterns of differentiation in a colour polymorphism and in neutral markers reveal rapid genetic changes in natural damselfly populations

The existence and mode of selection operating on heritable adaptive traits can be inferred by comparing population differentiation in neutral genetic variation between populations (often using F(ST) values) with the corresponding estimates for adaptive traits. Such comparisons indicate if selection acts in a diversifying way between populations, in which case differentiation in selected traits is expected to exceed differentiation in neutral markers [F(ST )(selected) > F(ST )(neutral)], or if negative frequency-dependent selection maintains genetic polymorphisms and pulls populations towards a common stable equilibrium [F(ST) (selected) < F(ST) (neutral)]. Here, we compared F(ST) values for putatively neutral data (obtained using amplified fragment length polymorphism) with estimates of differentiation in morph frequencies in the colour-polymorphic damselfly Ischnura elegans. We found that in the first year (2000), population differentiation in morph frequencies was significantly greater than differentiation in neutral loci, while in 2002 (only 2 years and 2 generations later), population differentiation in morph frequencies had decreased to a level significantly lower than differentiation in neutral loci. Genetic drift as an explanation for population differentiation in morph frequencies could thus be rejected in both years. These results indicate that the type and/or strength of selection on morph frequencies in this system can change substantially between years. We suggest that an approach to a common equilibrium morph frequency across all populations, driven by negative frequency-dependent selection, is the cause of these temporal changes. We conclude that inferences about selection obtained by comparing F(ST) values from neutral and adaptive genetic variation are most useful when spatial and temporal data are available from several populations and time points and when such information is combined with other ecological sources of data.     

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.     

Continuous monitoring of phytoplankton dynamics in Lake Balaton (Hungary) using on-line delayed fluorescence excitation spectroscopy

1. This study introduces delayed fluorescence (DF) excitation spectroscopy as an on‐line tool for in situ monitoring of the composition and biomass of various colour classes of phytoplankton when they are photosynthetically active (cyanobacteria, chlorophytes, chromophytes and cryptophytes). The DF data are validated by comparison with those from conventional methods (weekly microscopic counts and the measurement of chlorophyll concentration). 2. The composition of phytoplankton as assessed by DF agreed reasonably well with the results from microscopic counts, particularly when differences in chlorophyll‐specific DF integrals of the various colour classes were taken into account. 3. Integrals of DF spectra were converted into concentration of chlorophyll a using empirical factors derived from field data. The value of the conversion factor was nearly twice as high when the relative abundance of cyanobacteria was low (<15%) than when it was high. The converted DF‐chl time series agreed well with chlorophyll measurements particularly when blooms were developing. As the DF method is inherently free of the interference caused by pigment degradation products, the discrepancy between the two data sets increased during the collapse of blooms and when sediment resuspension was intense. 4. Fourier spectrum analysis of the time series of DF‐chl indicated that samples must be taken, at a minimum, every 2–3 days to capture the dynamics of phytoplankton. As a consequence, the dynamics of various algal blooms, including their timing, duration and net growth rate, could be estimated with greater confidence than by using conventional methods alone. 5. On‐line DF spectroscopy is an advanced technique for monitoring daily the biomass and composition of the photosynthetically active phytoplankton in aquatic environments, including turbid shallow lakes. At present, the detection limit is around 1 mg DF‐chl a m^?3 in terms of total biomass but confidence in estimates of phytoplankton composition declines sharply below about 5 mg chl a m^?3. 6. On‐line DF spectroscopy represents a promising approach for monitoring phytoplankton. It will be useful in water management where it can act as an early‐warning system of declines in water quality. In basic ecological research it can supplement manual methods. While default calibration spectra may be acceptable for routine monitoring, we suggest a careful individual calibration of the DF spectrometer for basic research. The statistical methods developed here help to assess the adequacy of various calibration sets.     

Dynamics of outgrowth in a continuum model of neurite elongation

Neurite outgrowth (dendrites and axons) should be a stable, but easily regulated process to enable a neuron to make its appropriate network connections during development. We explore the dynamics of outgrowth in a mathematical continuum model of neurite elongation. The model describes the construction of the internal microtubule cytoskeleton, which results from the production and transport of tubulin dimers and their assembly into microtubules at the growing neurite tip. Tubulin is assumed to be largely synthesised in the cell body from where it is transported by active mechanisms and by diffusion along the neurite. It is argued that this construction process is a fundamental limiting factor in neurite elongation. In the model, elongation is highly stable when tubulin transport is dominated by either active transport or diffusion, but oscillations in length may occur when both active transport and diffusion contribute. Autoregulation of tubulin production can eliminate these oscillations. In all cases a stable steady-state length is reached, provided there is intrinsic decay of tubulin. Small changes in growth parameters, such as the tubulin production rate, can lead to large changes in length. Thus cytoskeleton construction can be both stable and easily regulated, as seems necessary for neurite outgrowth during nervous system development. Action Editor: Upinder Bhalla     

Regulation of Rotifer Community by Predation of Cyclops vicinus (Copepoda) in the Římov Reservoir in Spring

The structuring role and predation impact of Cyclops vicinus on the rotifer community was studied using in situ enclosure experiments in Římov Reservoir during the spring of 2000. Seasonal changes in abundance and birth rates of the rotifer species Synchaeta lakowitziana , Polyarthra spp., Keratella cochlearis and Kellicottia longispina were related to predation pressure, i.e. selectivity and cropping rates of C. vicinus . All four rotifer species were found to be consumed by C. vicinus whose predation rates showed a marked succession through the spring period and corresponded to changes in the relative abundance of the pelagic species. The highest predation rates exhibited by C. vicinus were on Synchaeta lakowitziana and Polyarthra spp., being 11.3 and 6.4 ind Cyclops ^–1 day^–1 respectively. The activity of the predator switched among three types of preferred food during the spring period. The most preferred, Synchaeta, was replaced by Polyarthra after Synchaeta became extinct and these species were replaced by Keratella when both soft‐bodied forms were not available in sufficient quantities. Loricated species (K. cochlearis and K. longispina) were consumed at a slower rate. We calculated the proportion of rotifer production cropped day^–1 in order to estimate the predation impact of C. vicinus , which ranged from 11.6 to more than 100% over time and rotifer species. This shows that relative predation impact can be high not only on species most strongly selected, but also on species which are not selected. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.     

Stoichiometric and growth responses of a freshwater filamentous green alga to varying nutrient supplies: slow and steady wins the race

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Influence of Colophospermum mopane canopy cover on litter decomposition and nutrient dynamics in a semi‐arid African savannah

The objective of this study was to investigate the influence of mopane canopy cover on litter decomposition in a semi‐arid African savannah. We used a randomized block design with five blocks of 100 × 100 m demarcated in a 10‐ha pocket of open mopane woodland. Litterbags were placed beneath large (8.3 m crown diameter) and small mopane trees (2.7 m crown diameter) and in the intercanopy area. Decomposition was fastest in the intercanopy area exposed to solar radiation (k = 0.35 year^?1), intermediate beneath small trees (k = 0.28 year^?1) and slowest beneath large trees (k = 0.23 year^?1). Soil temperatures beneath small and large trees were 3–5 and 6–9°C lower than in the intercanopy area, respectively. Bacterial and fungal counts were significantly higher (P < 0.05) beneath large than small trees and in the intercanopy area. The amount of N and P released did not vary significantly among sampling sites. Soil moisture in the dry season was similar among sampling sites but rainy‐season soil moisture was significantly greater (P < 0.05) beneath large than small trees and in the intecanopy area. Mopane canopy cover retarded litter decomposition suggesting that photodegradation could be an important factor controlling carbon turnover in semi‐arid African savannahs.