Gareth R. Jones

Mobility of photosynthetic complexes in thylakoid membranes

The structures of many photosynthetic pigment–protein complexes have now been determined, but a real understanding of the photosynthetic membrane at the molecular level will also require knowledge of the organization and dynamics of these complexes in the intact membrane. Using fluorescence recovery after photobleaching (FRAP) and a scanning confocal microscope, we have made direct measurements in vivo of the lateral diffusion of light-harvesting complexes and reaction centres in the thylakoid membranes of the cyanobacterium Dactylococcopsis salina . We find that the phycobilisomes (the accessory light-harvesting complexes of cyanobacteria) diffuse quite rapidly, but that photosystem II is immobile on the timescale of the measurement, indicating that the linkage between phycobilisomes and photosystem II is unstable. We propose that the lateral diffusion of phycobilisomes is involved in regulation of photosynthetic light-harvesting (state 1–state 2 transitions). The mobility of the phycobilisomes may also be essential to allow the synthesis and repair of thylakoid membrane components.

Preformed Oligomeric Epidermal Growth Factor Receptors Undergo an Ectodomain Structure Change during Signaling

Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptors ectodomain as measured by FRET, show that 14% of EGFRs in A431 cells are oligomerized before growth factor binding. After binding growth factor and signaling, these oligomers dissociate before releasing growth factor. Time courses of the FRET-derived distances between constitutively oligomerized EGFRs during signal transduction show a transient structural change in the extracellular domain, which occurs simultaneously with the production of intracellular Ca2+ signals. The FRET measurements also show a slow increase in oligomerization of EGFR monomers after growth factor binding. The structural change found in the extracellular domain of oligomeric EGFRs is similar to that shown by others for EPO, Neu, Fas, and tumor necrosis factor receptors, and may therefore be a common property of the transduction of the receptor-mediated signals.

Synchrotron X-ray investigations into the lamellar gel phase formed in pharmaceutical creams prepared with cetrimide and fatty alcohols

Semisolid liquid paraffin-in-water emulsions (aqueous creams) prepared from cetrimide/fatty alcohol mixed emulsifiers, and ternary systems formed by dispersing the mixed emulsifier in controlled percentages of water were examined as they aged using a combination of low and high angle X-ray diffraction measurements (Daresbury Laboratory Synchrotron Radiation Source). The results were correlated with the rheological properties measured in earlier studies. The cationic emulsifying wax showed phenomenal swelling in water. The reflection that incorporates interlamellar water increased continuously from 74 A at 28% water to over 500 A at 93% water. The trend was not influenced by the method of incorporation of the components and swollen lamellar phase was also identified in the corresponding emulsion. The swelling, which was due to electrostatic repulsion, was suppressed by salt and was reduced when the surfactant counterion was changed from Br(-) to Cl(-). Changes in rheological properties on storage and in the presence of salt were correlated with changes in water layer thickness. High angle diffraction confirmed that the hydrocarbon bilayers were in the hexagonal alpha-crystalline mode of packing. Ternary systems and creams prepared from pure alcohols, although initially semisolid, were rheologically unstable and broke down. Low angle X-ray study into the kinetics of structure breakdown showed that the swollen lamellar gel phase formed initially swells even further on storage before separating.

CD12: a new high-flux beamline for ultraviolet and vacuum-ultraviolet circular dichroism on the SRS, Daresbury.

This paper describes the commissioning and characterization of an SRS bending-magnet beamline constructed for the measurement of vacuum-ultraviolet circular dichroism on biological and other materials. The beamline provides photon fluxes of many orders of magnitude greater than commercial instruments or beamlines at other synchrotron radiation facilities. The beamline uses the conventional approach of utilizing the plane polarized light emitted from the bending magnet which is subsequently converted into circularly polarized light using a photoelastic modulator with a switching frequency of 50 kHz. The beamline has a best wavelength resolution of 0.5 nm and stray light levels better than 0.01%. The latter may be predicted to give improved performance over other beamlines at synchrotron radiation sources especially when short-wavelength CD spectra are to be collected. An example spectrum and submillisecond time-resolved CD profile are given and the impact that the new beamline is likely to have is speculated on. The ultimate flux limitations of the technique with regard to the avoidance of the effects of radiation damage are also discussed.

Stabilizing nonpolar/polar side-chain interactions in the alpha-helix.

A simplistic, yet often used, view of protein stability is that amino acids attract other amino acids with similar polarity, whereas nonpolar and polar side chains repel. Here we show that nonpolar/polar interactions, namely Val or Ile bonding to Lys or Arg in α‐helices, can in fact be stabilizing. Residues spaced i, i + 4 in α‐helices are on the same face of the helix, with potential to favorably interact and stabilize the structure. We observe that the nonpolar/polar pairs Ile‐Lys, Ile‐Arg, and Val‐Lys occur in protein helices more often than expected when spaced i, i + 4. Partially helical peptides containing pairs of nonpolar/polar residues were synthesized. Controls with i, i + 5 spacing have the residues on opposite faces of the helix and are less helical than the test peptides with the i, i + 4 interactions. Experimental circular dichroism results were analyzed with helix‐coil theory to calculate the free energy for the interactions. All three stabilize the helix with ΔG between −0.14 and −0.32 kcal · mol−1. The interactions are hydrophobic with contacts between Val or Ile and the alkyl groups in Arg or Lys. Side chains such as Lys and Arg can thus interact favorably with both polar and nonpolar residues. Proteins 2001;45:449–455.

Homeoviscous adaptation under pressure: the pressure dependence of membrane order in brain myelin membranes of deep-sea fish

Steady-state and time-resolved anistropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence have been used to compare the hydrocarbon order of brain myelin membranes from a shallow water (plaice) and two deep-sea fish species (Coryphenoides rupestris and Coryphenoides armatus). At atmospheric pressure the deep sea fish displayed lower steady-state anisotropies than shallow water species although the pressure dependence of anisotropy was similar in all species. Time-resolved measurements allowed the separate determination of the rate of probe motion from the amplitude of that motion. Anisotropy decays were analysed in terms of two correlation times and a constant (r infinity). The r infinity and mean value of P2 order parameter for all species increased with pressure, the graphs for deep-sea species being translated to higher pressures relative to shallow-water species. The resulting pressure coefficients for C. armatus was distinctly less than for the two shallower species. These time-resolved studies show that the interspecific differences provide for similar order parameters in all three species when corrected to their respective habitat conditions of pressure and temperature. This indicates that myelin order is highly conserved despite the profound ordering effects of high hydrostatic pressure.

The conformation of calmodulin: A substantial environmentally sensitive helical transition in Ca4-calmodulin with potential mechanistic function

The conformation of Ca4‐calmodulin in solution, as assessed by far‐UV peptide circular dichroism, contains significantly less α‐helix than the proposed X‐ray crystal structure. We now show that Ca4‐calmodulin adopts significant additional helical structure in solution in the presence of a helicogenic solvent (50%, v/v, aqueous 2,2,2‐trifluoroethanol or 50%, v/v, methylpentane‐5,5‐diol). We suggest that the long continuous helix (residues 66–92 of the crystal structure)is not necessarily a normal feature of the calmodulin structure in solution, and may be due in part to the conditions of crystallisation. This result is supported by time‐resolved tyrosine fluorescence anisotropy studies indicating that Ca4‐calmodulin in solution is an essentially compact globular structure which undergoes isotropic rotational motion. We conclude that, under appropriate ionic and apolar environmental conditions, Ca4‐calmodulin undergoes a substantial helical transition, which may involve residues in the central region of the molecule. Such a transition could have an important function in determining specificity and affinity in interactions of calmodulin with different target sequences of Ca2+‐dependent regulatory enzymes.

Applications of extended ultra-violet circular dichroism spectroscopy in biology and medicine

Deep ultra-violet circular dichroism is fast becoming an important technique in structural biology. The exponential increase in the number of protein structures deposited in the Protein Data Bank together with programs that extract protein secondary structure from atomic coordinates and the advancement of the software to analyse circular dichroic spectra, have revolutionised the technique. In addition, the extended short wavelength data afforded by synchrotron radiation is set to have a major impact on the development of the area. We have selected three diverse areas of research and development in the biomedical sciences to illustrate the ubiquity of the technique for future applications in the area of biomedical research. For example, the high flux of synchrotron radiation has provided a gold standard for the assay of the lipoprotein HDL in serum which has been proven to reverse the effects of coronary heart disease. In a second example, the high flux of synchrotron radiation enables the recording of millisecond data during the conformational changes in proteins over their spectrum, mapping out changes to protein secondary structure and thus providing absolute structural measurements in the millisecond time regime. In the third example, subtle conformational changes are interpreted from the extended CD spectra on protein–drug binding, distinguishing between induced binding effects and the conformational changes in the target protein. The strengths and weaknesses of extended ultra-violet circular dichroism using synchrotron radiation are discussed using these examples as a template.

Subnanosecond polarized microfluorimetry in the time domain: An instrument for studying receptor trafficking in live cells

We describe an instrument designed to monitor molecular motions in multiphasic, weakly fluorescent microscopic systems. It combines synchrotron radiation, a low irradiance polarized microfluorimeter, and an automated, multiframing, single-photon-counting data acquisition system, and is capable of continually accumulating subnanosecond resolved anisotropy decays with a real-time resolution of about 60 s. The instrument has initially been built to monitor ligand–receptor interactions in living cells, but can equally be applied to the continual measurement of any dynamic process involving fluorescent molecules, that occurs over a time scale from a few minutes to several hours. As a particularly demanding demonstration of its capabilities, we have used it to monitor the environmental constraints imposed on the peptide hormone epidermal growth factor during its endocytosis and recycling to the cell surface in live cells.

A high sensitivity time‐resolved microfluorimeter for real‐time cell biology

We describe an instrument based on the novel combination of synchrotron radiation, a high sensitivity time‐resolved microfluorimeter, and a multiframe single photon counting data acquisition system. This instrument has been designed specifically to measure kinetic events in live cells using fluorescence resonance energy transfer, and is capable of rapidly collecting multiple consecutive decay profiles from a small number of fluorophores. The low irradiance on the samples (<10 mW/cm2) greatly reduces probe photobleaching and specimen photodamage during prolonged exposures. The Daresbury Synchrotron Radiation Source provides fully wavelength tunable light pulses that have a full width half‐maximum of 160 ps at a repetition rate of 3.125 MHz, with the high temporal stability required for continuous measurements over periods of hours. A very low limit of detection (<104 molecules/mW/cm2) is accomplished by combining a high‐gain single photon counting detection system with a low fluorescence background optical...