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Sol M Gruner

Publications and source records attributed to Sol M Gruner.

18 recordsLinked to original sources
PubMed2006

The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring.

The KtrAB ion transporter is a complex of the KtrB membrane protein and KtrA, an RCK domain. RCK domains regulate eukaryotic and prokaryotic membrane proteins involved in K(+) transport. Conflicting functional models have proposed two different oligomeric arrangements for RCK domains, tetramer versus octamer. Our results for the KtrAB RCK domain clearly show an octamer in solution and in the crystal. We determined the structure of this protein in three different octameric ring conformations that resemble the RCK-domain octamer observed in the MthK potassium channel but show striking differences in size and symmetry. We present experimental evidence for the association between one RCK octameric ring and two KtrB membrane proteins. These results provide insights into the quaternary organization of the KtrAB transporter and its mechanism of activation and show that the RCK-domain octameric ring model is generally applicable to other ion-transport systems.

Bacteria↗
PubMed2006

Solution of protein crystallographic structures by high-pressure cryocooling and noble-gas phasing.

Room-pressure flash-cryocooling of protein crystals is the standard way to reduce radiation damage during data collection. Typically, it is necessary to find cryoprotection conditions by trial and error, a process that is not always successful. Recently, a new method, high-pressure cryocooling, was developed that does not require penetrative cryoprotectants and typically yields very high quality diffraction. Since this method involves helium gas as a pressurizing medium, it was of great interest to see whether the method could be extended to diffraction phasing by the incorporation of heavy noble gases such as krypton. A modified Kr-He high-pressure cyrocooling procedure is described wherein crystals are first pressurized with krypton gas to 10 MPa for 1 h. The krypton pressure is then released and the crystals are repressurized with helium over 150 MPa and cooled to liquid-nitrogen temperatures. Porcine pancreas elastase (PPE; 240 residues, 26 kDa) was selected as a test case for this study. Excellent diffraction was achieved by high-pressure cryocooling without penetrating cryoprotectants. A single 0.31 occupied krypton site in a PPE molecule [Bijvoet amplitude ratio (|DeltaF|/F) of 0.53%] was successfully used for SAD phasing at 1.3 A. This method has the potential to greatly simplify obtaining protein structures.

Air Pressure↗
PubMed2006

Multilayer X-ray optics at CHESS.

Almost half of the X-ray beamlines at the Cornell High Energy Synchrotron Source (CHESS) are based on multilayer optics. ;Traditional' multilayers with an energy resolution of DeltaE/E approximately 2% are routinely used to deliver X-ray flux enhanced by a factor of 10(2) in comparison with standard Si(111) optics. Sagittal-focusing multilayers with fixed radius provide an additional factor of 10 gain in flux density. High-resolution multilayer optics with DeltaE/E approximately 0.2% are now routinely used by MacCHESS crystallographers. New wide-bandpass multilayers with DeltaE/E = 5% and 10% have been designed and tested for potential applications in macromolecular crystallography. Small d-spacing multilayers with d < or = 20 A have been successfully used to extend the energy range of multilayer optics. Analysis of the main characteristics of the Mo/B4C and W/B4C small d-spacing multilayer optics shows enhancement in their performance at higher energies. Chemical vapour deposited SiC, with a bulk thermal conductivity of a factor of two higher than that of silicon, has been successfully introduced as a substrate material for multilayer optics. Characteristics of different types of multilayer optics at CHESS beamlines and their applications in a variety of scattering, diffraction and imaging techniques are discussed.

Crystallography, X-Ray↗
PubMed2005

Cooperative water filling of a nonpolar protein cavity observed by high-pressure crystallography and simulation.

Formation of a water-expelling nonpolar core is the paradigm of protein folding and stability. Although experiment largely confirms this picture, water buried in "hydrophobic" cavities is required for the function of some proteins. Hydration of the protein core has also been suggested as the mechanism of pressure-induced unfolding. We therefore are led to ask whether even the most nonpolar protein core is truly hydrophobic (i.e., water-repelling). To answer this question we probed the hydration of an approximately 160-A(3), highly hydrophobic cavity created by mutation in T4 lysozyme by using high-pressure crystallography and molecular dynamics simulation. We show that application of modest pressure causes approximately four water molecules to enter the cavity while the protein itself remains essentially unchanged. The highly cooperative filling is primarily due to a small change in bulk water activity, which implies that changing solvent conditions or, equivalently, cavity polarity can dramatically affect interior hydration of proteins and thereby influence both protein activity and folding.

Crystallography↗
PubMed2005

High-pressure cooling of protein crystals without cryoprotectants.

Flash-cooling of protein crystals is the best known method to effectively mitigate radiation damage in macromolecular crystallography. To prevent physical damage to crystals upon cooling, suitable cryoprotectants must usually be found, a process that is time-consuming and in some cases unsuccessful. A method is described to cool protein crystals in high-pressure helium gas without the need for penetrative cryoprotectants. The method involves mounting protein crystals from the native mother liquor in a cryoloop with a droplet of oil, pressurizing the crystal to 200 MPa in He gas, cooling the crystal under pressure and then releasing the pressure. The crystal is then removed from the apparatus under liquid nitrogen and handled thereafter like a normal cryocooled crystal. Results are presented from three representative proteins. Dramatic improvement in diffraction quality in terms of resolution and mosaicity was observed in all cases. A mechanism for the pressure cooling is proposed involving high-density amorphous (HDA) ice which is produced at high pressure and is metastable at room pressure and 110 K.

Agkistrodon↗
PubMed2005

Crystallographic data collection using a 0.22% bandwidth multilayer.

To bridge the gap between traditional multilayer and crystal optics a high-resolution multilayer monochromator with a bandwidth of 0.22% has been designed and installed on a bending-magnet beamline (F3) at the Cornell High Energy Synchrotron Source (CHESS) to provide an unfocused monochromatic X-ray beam for protein crystallography experiments. Crystallographic data of excellent quality from a medium-sized protein, Concanavalin A, were collected and processed using standard crystallographic programs. The data were successfully used for a structure solution and refinement. The flux from the multilayer monochromator is enhanced, relative to that from a flat Si(111) monochromator, by a factor of 5; consequently, data collection is faster and/or smaller samples may be used. At the same time, the bandwidth is narrow enough to avoid streaked spots. This experiment suggests that multilayer optics may play a valuable role in satisfying the demands of the structural biology community for rapid X-ray data collection, particularly at under-utilized bending-magnet beamlines.

Concanavalin A↗
PubMed2005

Formation of homogeneous unilamellar liposomes from an interdigitated matrix.

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.

Ethanol↗
PubMed2004

Diastereoselective alkylation of beta-amino esters: structural and rate studies reveal alkylations of hexameric lithium enolates.

Alkylation of beta-amino ester enolates proceeds with high diastereoselectivity. Single crystal, powder, and solution X-ray diffraction studies of the enolate show that the racemic enolate forms prismatic hexamers. 6Li NMR spectroscopic studies on partially racemic enolates reveal complex mixtures of homo- and heterochiral hexamers. An implicit fit of the aggregate populations to the Boltzmann distribution provides the free energy differences and equilibrium constants for the ensemble. Rate studies show that enolate alkylation occurs directly from the hexamer with participation by THF. A mechanism based on the alkylation of a ladder-like aggregate is proposed.

Alkylation↗
PubMed2004

Characterization of beta-amino ester enolates as hexamers via 6Li NMR spectroscopy.

Low-temperature 6Li NMR spectroscopic studies on a chiral beta-amino ester enolate reveal a complex mixture of homo- and heterochiral aggregates. Subsequent warming of the samples led to rapid intra-aggregate exchange, resulting in four distinct resonances consistent with an ensemble of hexamers. An implicit fit of the aggregate populations to the Boltzmann distribution provided the free energy differences and equilibrium constants. An X-ray crystal structure obtained from the racemic enolate is consistent with the predominant aggregate in solution.

Alkylation↗
PubMed2004

Nanohybrids from liquid crystalline extended amphiphilic dendrimers.

A novel extended amphiphilic dendrimer with linear poly(ethylene oxide) (PEO) attached to a PEO-like dendritic core as hydrophilic fraction and eight docosyl chain branches as hydrophobic fraction has been prepared for the use as structure-directing agent for silica-type materials. The extended dendrimer exhibits a hexagonal columnar liquid crystalline phase in the melt. Organically modified inorganic precursors and the extended dendrimer co-assemble into nanostructured hybrids. Hybrids with 0.44 weight fraction (fw) of aluminosilicate show a lamellar morphology, while hybrids with 0.21 fw exhibit a cylindrical structure. Nanostructures were characterized by a combination of small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The results suggest that dendrimer-based amphiphiles may provide an exciting platform for the formation of multifunctional organic-inorganic nanostructured hybrid materials with unique structural characteristics.

Journal Article↗
PubMed2003

The plumber's nightmare: a new morphology in block copolymer-ceramic nanocomposites and mesoporous aluminosilicates.

A novel cubic bicontinuous morphology is found in polymer-ceramic nanocomposites and mesoporous aluminosilicates that are derived by an amphiphilic diblock copolymer, poly(isoprene-b-ethylene oxide) (PI-b-PEO), used as a structure-directing agent for an inorganic aluminosilicate. Small-angle X-ray scattering (SAXS) was employed to unambiguously identify the Im(-)3m crystallographic symmetry of the materials by fitting individual Bragg peak positions in the two-dimensional X-ray images. Structure factor calculations, in conjunction with results from transmission electron microscopy, were used to narrow the range of possible structures consistent with the symmetry and showed the plumber's nightmare morphology to be consistent with the data. The samples are made by deposition onto a substrate that imposes a strain field, generating a lattice distortion. This distortion is quantitatively analyzed and shown to have resulted in shrinkage of the crystallites by approximately one-third in a direction perpendicular to the substrate, in both as-made composites and calcined ceramic materials. Finally, the observation of the bicontinuous block-copolymer-derived hybrid morphology is discussed in the context of a pseudo-ternary morphology diagram and compared to existing studies of ternary phase diagrams of amphiphiles in a mixture of two solvents. The calcined mesoporous materials have potential applications in the fields of catalysis, separation technology, and microelectronics.

Aluminum Silicates↗
PubMed2002

Rapid compaction during RNA folding.

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

Animals↗
PubMed2002

X-ray imaging of shock waves generated by high-pressure fuel sprays.

Synchrotron x-radiography and a fast x-ray detector were used to record the time evolution of the transient fuel sprays from a high-pressure injector. A succession of 5.1-microsecond radiographs captured the propagation of the spray-induced shock waves in a gaseous medium and revealed the complex nature of the spray hydrodynamics. The monochromatic x-radiographs also allow quantitative analysis of the shock waves that has been difficult if not impossible with optical imaging. Under injection conditions similar to those found in operating engines, the fuel jets can exceed supersonic speeds and result in gaseous shock waves.

Cerium↗
PubMed2002

Determination of L(alpha)-H(II) phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine.

The thermodynamic properties of fully-hydrated lipids provide important information about the stability of membranes and the energetic interactions of lipid bilayers with membrane proteins (Nagle and Scott, Physics Today, 2:39, 1978). The lamellar/inverse hexagonal (L(alpha)-H(II)) phase transition of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) water mixtures is a first-order transition and, therefore, at constant pressure, must have a thermodynamically well-defined equilibrium transition temperature. The observed transition temperature is known to be dependent upon the rate at which the temperature is changed, which accounts for the many different values in the literature. X-ray diffraction was used to study the phase transition of fully-hydrated DOPE to determine the rate-independent transition temperature, T(LH). Samples were heated or cooled for a range of rates, 0.212 < r < 225 degrees C/hr, and the rate-dependent apparent phase transition temperatures, T(A)(r) were determined from the x-ray data. By use of a model-free extrapolation method, the transition temperature was found to be T(LH) = 3.33 +/- 0.16 degrees C. The hysteresis, /T(A)(r) - T(LH)/, was identical for heating and cooling rates, +/-r, and varied as /r/beta for beta approximately 1/4. This unexpected power-law relationship is consistent with a previous study (Tate et al., Biochemistry, 31:1081-1092, 1992) but differs markedly from the exponential behavior of activation barrier kinetics. The methods used in this study are general and provide a simple way to determine the true mesomorphic phase transition temperatures of other lipid and lyotropic systems.

Hot Temperature↗
PubMed2002

Probing substates in sperm whale myoglobin using high-pressure crystallography.

Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.

Animals↗