Divalent Cations and the Divergence of ßγ-Crystallin Function.

The ßγ-crystallin superfamily contains both ß- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate ßγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial ßγ-crystallins bind calcium ions. The ßγ-crystallin from the tunicate Ciona intestinalis (Ci-ßγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-ßγ is valuable for investigating the evolution of the ßγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-ßγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-ßγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-ßγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.

Kinetics of Trifluoromethane Clathrate Hydrate Formation from CHF3 Gas and Ice Particles.

We report the formation kinetics of trifluoromethane clathrate hydrate (CH) from less than 75 µm diameter ice particles and CHF3 gas. As previously observed for difluoromethane and propane hydrate formation, the initial stages of the reaction exhibit a strong negative correlation of the reaction rate with temperature, consistent with a negative activation energy of formation. The values obtained for trifluoromethane, ca. -6 kJ/mol (H2O), are similar to those for difluoromethane, even though the two molecules have different intermolecular interactions and sizes. The activation energy is lesser per mole of H2O, but greater per mole of guest molecule, than for propane hydrate, which has a different crystal structure. We propose a possible explanation for the negative activation barrier based on the stabilization of metastable structures at low temperature. A pronounced dependence of the formation kinetics on the gas flow rate into the cell is observed. At 253 K and a flow rate of 15 mmol/h, the stage II enclathration of trifluoromethane proceeds so quickly that the overpressure, the difference between the gas cell pressure and the hydrate vapor pressure, is only 0.06 MPa.

Calcium Binding Dramatically Stabilizes an Ancestral Crystallin Fold in Tunicate ßγ-Crystallin.

The tunicate (Ciona intestinalis) ßγ-crystallin represents an intermediate case between the calcium-binding proteins ancestral to the vertebrate ßγ-crystallin fold and the vertebrate structural crystallins. Unlike the structural ßγ-crystallins in the vertebrate eye lens, this ßγ-crystallin strongly binds Ca2+. Furthermore, Ca2+ binding greatly stabilizes the protein, an effect that has previously been observed in microbial ßγ-crystallins but not in those of vertebrates. This relationship between binding and protein stabilization makes the tunicate ßγ-crystallin an interesting model for studying the evolution of the human ßγ-crystallin. We also compare and contrast the binding sites of tunicate ßγ-crystallin with those of other ßγ-crystallins to develop hypotheses about the functional origin of the lack of Ca2+-binding sites in human crystallins.

In situ NMR measurement of macromolecule-bound metal ion concentrations.

Many nucleic acids and proteins require divalent metal ions such as Mg(2+) and Ca(2+) for folding and function. The lipophilic alignment media frequently used as membrane mimetics also bind these divalent metals. Here we demonstrate the use of (31)P NMR spectrum of a metal ion chelator (deoxycytidine diphosphate) to measure the bound [Mg(2+)] and [Ca(2+)] in situ for several biological model systems at relatively high divalent ion concentrations (1-10 mM). This method represents a general approach to measuring divalent metal ion binding in NMR samples where the amount and type of metal ion added to the system is known.

Prion nucleation site unmasked by transient interaction with phospholipid cofactor.

Infectious mammalian prions can be formed de novo from purified recombinant prion protein (PrP) substrate through a pathway that requires the sequential addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA cofactor molecules. Recent studies show that the initial interaction between PrP and POPG causes widespread and persistent conformational changes to form an insoluble intermediate species, termed PrP(Int1). Here, we characterize the mechanism and functional consequences of the interaction between POPG and PrP. Negative-stain electron microscopy of PrP(Int1) revealed the presence of amorphous aggregates. Pull-down and photoaffinity label experiments indicate that POPG induces the formation of a PrP(C) polybasic-domain-binding neoepitope within PrP(Int1). The ongoing presence of POPG is not required to maintain PrP(Int1) structure, as indicated by the absence of stoichiometric levels of POPG in solid-state NMR measurements of PrP(Int1). Together, these results show that a transient interaction with POPG cofactor unmasks a PrP(C) binding site, leading to PrP(Int1) aggregation.

CSA-enabled spin diffusion leads to MAS rate-dependent T1's at high field.

A surprisingly strong spin rate dependence of (15)N and (13)C NMR T(1) times in magic angle spinning experiments on solid peptides is demonstrated. Using a variety of isotopomers, the phenomenon is shown to be the result of chemical shift anisotropy-mediated spin diffusion. This effect has the potential to be used to detect long-range distance constraints in macromolecular systems.

Design and construction of a quadruple-resonance MAS NMR probe for investigation of extensively deuterated biomolecules.

Extensive deuteration is frequently used in solid-state NMR studies of biomolecules because it dramatically reduces both homonuclear (1H-1H) and heteronuclear (1H-13C and 1H-15N) dipolar interactions. This approach greatly improves resolution, enables low-power rf decoupling, and facilitates 1H-detected experiments even in rigid solids at moderate MAS rates. However, the resolution enhancement is obtained at some cost due the reduced abundance of protons available for polarization transfer. Although deuterium is a useful spin-1 NMR nucleus, in typical experiments the deuterons are not directly utilized because the available probes are usually triple-tuned to 1H,13C and 15N. Here we describe a 1H/13C/2H/15N MAS ssNMR probe designed for solid-state NMR of extensively deuterated biomolecules. The probe utilizes coaxial coils, with a modified Alderman-Grant resonator for the 1H channel, and a multiply resonant solenoid for 13C/2H/15N. A coaxial tuning-tube design is used for all four channels in order to efficiently utilize the constrained physical space available inside the magnet bore. Isolation among the channels is likewise achieved using short, adjustable transmission line elements. We present benchmarks illustrating the tuning of each channel and isolation among them and the magnetic field profiles at each frequency of interest. Finally, representative NMR data are shown demonstrating the performance of both the detection and decoupling circuits.

Integrated kinetics for the production of glucose in plant cells and the effect of temperature.

We prepare a temperature-dependent formulation of the integrated kinetics for the overall process of photosynthesis in eukaryotic cells. To avoid complexity, the C4 plants are chosen because their rate of photosynthesis is independent of the partial pressure of O2. A systematically simplified but comprehensive scheme for both light and dark reactions is considered. The reaction rate per reaction center in the thylakoid membrane is related to the rate of exciton transfer between chlorophyll neighbors. An expression is formulated for the light reaction rate (R1'). The NADPH formation rate is related to R1' and the survival probability of the membrane. Rates of different steps in the simplified scheme can be related to each other by applying a few steady state conditions. The saturation probability of CO2 in a bundle sheath is also considered. The photochemical efficiency (phi) appears in terms of these probabilities. We find the glucose production rate as R(glucose) = (8/3) upsilon L: [corrected] R1'phi g(T)([G3P]/[P(i)]2) exp(-deltaG(E)S/RT), where g(T) is the activation quotient of the involved enzymes, G3P and P(i) represent glyceraldehyde-3-phosphate and inorganic phosphates, and deltaG(E)S is the free energy for the apparent equilibrium between G3P and glucose. This is the first time that such a comprehensive expression for R(glucose) has been derived. The probabilities are generally given by sigmoid curves. The corresponding parameters can be easily determined. The quotient g(T) incorporates a Gaussian distribution for temperature dependence and a sigmoid function describing deactivation. The theoretical plots of photochemical efficiency and glucose production rate versus temperature are in excellent agreement with the experimental ones, thereby validating the formalism.

Propane Clathrate Hydrate Formation Accelerated by Methanol.

The role of methanol as both an inhibitor and a catalyst for the formation of clathrate hydrates (CHs) has been a topic of intense study. We report a new quantitative study of the kinetics of propane CH formation at 253 K from the reaction of propane gas with <75 µm ice particles that have been doped with varying amounts of methanol. We find that methanol significantly accelerates the formation reaction with quite small doping quantities. Even for only 1 methanol molecule per 10 000 water molecules, the maximum uptake rate of propane into CHs is enhanced and the initiation pressure is reduced. These results enable more efficient production of CHs for gas storage. This remarkable acceleration of the CH formation reaction by small quantities of methanol may place constraints on the mechanism of the inhibition effect observed under other conditions, usually employing much larger quantities of methanol.

Exploring Dynamics and Cage-Guest Interactions in Clathrate Hydrates Using Solid-State NMR.

Interactions between guest molecules and the water cages in clathrates are dominated by isotropic van der Waals forces at low temperatures because the cage structures satisfy the hydrogen bonding propensity of the water. However, above 200 K the water molecules become more labile and may interact strongly with the guests through hydrogen bonding. In this work we compare the dynamics of tetrahydrofuran (THF) and cyclopentane (CP) guests in the hydrate cages above 200 K. Since THF can form hydrogen bonds while CP cannot, the dynamics provide insight into host-guest hydrogen bonding. We use magic angle spinning (MAS) solid-state NMR to measure proton spin-lattice relaxation times (T1) of the guests as a function of temperature and find that the activation barrier to the motion of THF molecules is 4.7 kcal/mol (19.7 kJ/mol) at temperatures above 200 K. This is almost 5 times higher than the barrier at lower temperatures. In contrast, the barrier to guest motion in CP hydrate is found to be about 0.67 kcal/mol (2.8 kJ/mol), which agrees well with data at lower temperatures. These results demonstrate that hydrogen bonding interactions between the THF guest and the clathrate cage are significant above 200 K due to the host lattice mobility.

Accelerating multidimensional NMR and MRI experiments using iterated maps.

Techniques that accelerate data acquisition without sacrificing the advantages of fast Fourier transform (FFT) reconstruction could benefit a wide variety of magnetic resonance experiments. Here we discuss an approach for reconstructing multidimensional nuclear magnetic resonance (NMR) spectra and MR images from sparsely-sampled time domain data, by way of iterated maps. This method exploits the computational speed of the FFT algorithm and is done in a deterministic way, by reformulating any a priori knowledge or constraints into projections, and then iterating. In this paper we explain the motivation behind this approach, the formulation of the specific projections, the benefits of using a 'QUasi-Even Sampling, plus jiTter' (QUEST) sampling schedule, and various methods for handling noise. Applying the iterated maps method to real 2D NMR and 3D MRI of solids data, we show that it is flexible and robust enough to handle large data sets with significant noise and artifacts.