Biomedical subjects

Martin Caffrey

Publications and source records attributed to Martin Caffrey.

9 recordsLinked to original sources

PubMed2003

Specific radiation damage can be used to solve macromolecular crystal structures.

The use of third generation synchrotron sources has led to renewed concern about the effect of ionizing radiation on crystalline biological samples. In general, the problem is seen as one to be avoided. However, in this paper, it is shown that, far from being a hindrance to successful structure determination, radiation damage provides an opportunity for phasing macromolecular structures. This is successfully demonstrated for both a protein and an oligonucleotide, by way of which complete models were built automatically. The possibility that, through the exploitation of radiation damage, the phase problem could become less of a barrier to macromolecular crystal structure determination is discussed.

Crystallography, X-Ray↗

PubMed2003

Membrane protein crystallization.

The need for high-resolution structure information on membrane proteins is immediate and growing. Currently, the only reliable way to get it is crystallographically. The rate-limiting step from protein to structure is crystal production. An overview of the current ideas and experimental approaches prevailing in the area of membrane protein crystallization is presented. The long-established surfactant-based method has been reviewed extensively and is not examined in detail here. The focus instead is on the latest methods, all of which exploit the spontaneous self-assembling properties of lipids and detergent as vesicles (vesicle-fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic-phase method). In the belief that a knowledge of the underlying phase science is integral to understanding the molecular basis of these assorted crystallization strategies, the article begins with a brief primer on lipids, mesophases, and phase science, and the related issue of form and function as applied to lipids is addressed. The experimental challenges associated with and the solutions for procuring adequate amounts of homogeneous membrane proteins, or parts thereof, are examined. The cubic-phase method is described from the following perspectives: how it is done in practice, its general applicability and successes to date, and the nature of the mesophases integral to the process. Practical aspects of the method are examined with regard to salt, detergent, and screen solution effects; crystallization at low temperatures; tailoring the cubic phase to suit the target protein; different cubic-phase types; dealing with low-protein samples, colorless proteins, microcrystals, and radiation damage; transport within the cubic phase for drug design, cofactor retention, and phasing; using spectroscopy for quality control; harvesting crystals; and miniaturization and robotization for high-throughput screening. The section ends with a hypothesis for nucleation and growth of membrane protein crystals in meso. Thus far, the bicelle and vesicle-fusion methods have produced crystals of one membrane protein, bacteriorhodopsin. The experimental details of both methods are reviewed and their general applicability in the future is commented on. The three new methods are rationalized by analogy to crystallization in microgravity and with respect to epitaxy. A list of Web resources in the area of membrane protein crystallogenesis is included.

Crystallization↗

PubMed2002

Too hot to handle? Synchrotron X-ray damage of lipid membranes and mesophases.

The call for brighter synchrotron X-radiation sources for use in structural biology research is barely audible as we enter the new millennium. Our brightest sources are already creating havoc when used at design specifications because of radiation damage. The time is long overdue to take stock of where we are and where we wish to go with regards to using existing sources and to designing new ones. The problem of radiation damage is particularly acute in studies involving kinetics and mechanisms where cryo-techniques are not always viable. Accordingly, we need to understand the very nature of radiation damage and to devise means of minimizing it. This is the thrust of the current report as applied to lipid membranes and mesophases. The experiments were performed at the most brilliant beamlines at CHESS, the APS and the ESRF. Two very different types of radiation damage are reported here. One involves a dramatic phase transformation and the other a disordering of lamellar stacking. How beam energy and dose rate affect damage is also discussed. The work highlights the free-radical-mediated nature of the damage process and the need for additional studies if the most efficient use is to be made of an important resource, synchrotron radiation.

Animals↗

PubMed2002

Unit-cell volume change as a metric of radiation damage in crystals of macromolecules.

The use of third-generation synchrotron sources has led to renewed interest in the effect that ionizing radiation has on crystalline biological materials. Simple criteria have been sought to study the effects systematically. The unit-cell volume of protein crystals shows a linear increase with absorbed dose and has therefore been proposed to be such a measure. This paper demonstrates that the increase is sample dependent, and thus it might not be a useful indicator when comparing different samples. For individual samples, however, the increase can be used to quantify ambient temperature and dose-rate effects. In this study, highly absorbing cubic crystals of holoferritin have been used to accurately determine how cell volume changes with absorbed dose. The experiments show that, for this protein, a dose-rate effect exists and that trapped radicals can be mobilized at ca 180 K.

Crystallization↗

PubMed2002

Controlling membrane cholesterol content. A role for polyunsaturated (docosahexaenoate) phospholipids.

The molecular organization of cholesterol in 1,2-didocosahexaenoylphosphatidylcholine (22:6-22:6PC) and 1-stearoyl-2-docosahexaenoylphosphatidylcholine (18:0-22:6PC) bilayers was investigated. Using low- and wide-angle X-ray diffraction (XRD), we determined that the solubility of the sterol at 20 degrees C was 11 +/- 3 mol % in 22:6-22:6PC vs 55 +/- 3 mol % in 18:0-22:6PC bilayers. Solubility in the dipolyunsaturated membrane rose to 17 +/- 3 mol % at 40 degrees C, while in the saturated-polyunsaturated membrane there was no change within experimental uncertainty. We compared the molecular orientation of [3alpha-(2)H(1)]cholesterol incorporated into 22:6-22:6PC bilayers to its solubility limit and into 18:0-22:6PC bilayers to a comparable concentration (10 mol %) in solid-state (2)H NMR experiments. The sterol possessed a tilt angle alpha(0) = 24 degrees +/- 1 degrees in 22:6-22:6PC that was independent of temperature over a range from 20 to 40 degrees C. In contrast, the value was alpha(0) = 21 degrees +/- 1 degrees in 18:0-22:6 bilayers at 20 degrees C and increased to alpha(0) = 24 degrees +/- 1 degrees at 40 degrees C. We attribute the low solubility of cholesterol in 22:6-22:6PC membranes to steric incompatibility between the rigid steroid moiety and the highly disordered docosahexaenoic acid (DHA) chain, which has the potential to promote lateral heterogeneity within DHA-rich membranes. Considering 22:6-22:6PC to be the most unsaturated phospholipid found in vivo, this model membrane study provides a point of reference for elucidating the role of sterol-lipid interactions in controlling local compositional organization. Our results form the basis for a model that is consistent with cholesterol's ability to modulate the activity of certain neural transmembrane proteins.

Cholesterol↗

PubMed2002

Membrane protein crystallization in meso: lipid type-tailoring of the cubic phase.

Hydrated monoolein forms the cubic-Pn3m mesophase that has been used for in meso crystallization of membrane proteins. The crystals have subsequently provided high-resolution structures by crystallographic means. It is possible that the hosting cubic phase created by monoolein alone, which itself is not a common membrane component, will limit the range of membrane proteins crystallizable by the in meso method. With a view to expanding the range of applicability of the method, we investigated by x-ray diffraction the degree to which the reference cubic-Pn3m phase formed by hydrated monoolein could be modified by other lipid types. These included phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, cardiolipin, lyso-PC, a polyethylene glycol-lipid, 2-monoolein, oleamide, and cholesterol. The results show that all nine lipids were accommodated in the cubic phase to some extent without altering phase identity. The positional isomer, 2-monoolein, was tolerated to the highest level. The least well tolerated were the anionic lipids, followed by lyso-PC. The others were accommodated to the extent of 20-25 mol %. Beyond a certain concentration limit, the lipid additives either triggered one or a series of phase transitions or saturated the phase and separated out as crystals, as seen with oleamide and cholesterol. The series of phases observed and their order of appearance were consistent with expectations in terms of interfacial curvature changes. The changes in phase type and microstructure have been rationalized on the basis of lipid molecular shape, interfacial curvature, and chain packing energy. The data should prove useful in the rational design of cubic phase crystallization matrices with different lipid profiles that match the needs of a greater range of membrane proteins.

Cardiolipins↗

PubMed2002

Molecular organization of cholesterol in polyunsaturated membranes: microdomain formation.

The molecular organization of cholesterol in phospholipid bilayers composed of 1,2-diarachidonylphosphatidylcholine (20:4-20:4PC), 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4PC), and 20:4-20:4PC/18:0-20:4PC (1/1 mol) was investigated by solid-state (2)H NMR and by low- and wide-angle x-ray diffraction (XRD). On the basis of distinct quadrupolar powder patterns arising from [3 alpha-(2)H(1)]cholesterol intercalated into the membrane and phase separated as solid, solubility chi(NMR)(chol) = 17 +/- 2 mol% and tilt angle alpha(0) = 25 +/- 1 degrees in 20:4-20:4PC were determined. The corresponding values in 18:0-20:4PC were chi (NMR)(chol) > or = 50 mol% and alpha(0) = 16 +/- 1 degrees. Cholesterol solubility determined by XRD was chi(NMR)(chol) = 15 +/- 2 mol% and chi(NMR)(chol) = 49 +/- 1 mol% for 20:4-20:4PC and 18:0-20:4PC, respectively. XRD experiments show that the solid sterol is monohydrate crystals presumably residing outside the bilayer. The (2)H NMR spectrum for equimolar [3 alpha-(2)H(1)]cholesterol added to mixed 20:4-20:4PC/18:0-20:4PC (1/1 mol) membranes is consistent with segregation of cholesterol into 20:4-20:4PC and 18:0-20:4PC microdomains of <160 A in size that preserve the molecular organization of sterol in the individual phospholipid constituents. Our results demonstrate unambiguously that cholesterol has low affinity to polyunsaturated fatty acids and support hypotheses of lateral phase separation of membrane constituents into sterol-poor/polyunsaturated fatty acid-rich and sterol-rich/saturated fatty acid-rich microdomains.

Cholesterol↗

PubMed2002

Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection.

3T3 Cells↗

PubMed2002

An index of lipid phase diagrams.

There is a growing awareness of the utility of lipid phase behavior data in studies of membrane-related phenomena. Such miscibility information is commonly reported in the form of temperature-composition (T-C) phase diagrams. The current index is a conduit to the relevant literature. It lists lipid phase diagrams, their components and conditions of measurement, and complete bibliographic information. The main focus of the index is on lipids of membrane origin where water is the dispersing medium. However, it also includes records on acylglycerols, fatty acids, cationic lipids, and detergent-containing systems. The miscibility of synthetic and natural lipids with other lipids, with water, and with biomolecules (proteins, nucleic acids, carbohydrates, etc.) and non-biological materials (drugs, anesthetics, organic solvents, etc.) is within the purview of the index. There are 2188 phase diagram records in the index, the bulk (81%) of which refers to binary (two-component) T-C phase diagrams. The remainder is made up of more complex (ternary, quaternary) systems, pressure-T phase diagrams, and other more exotic miscibility studies. The index covers the period from 1965 through to July, 2001.

Lipids↗