Jane M. Vanderkooi

Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phophoslipid artificial membranes.

The fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles is a function of the physical state of the lipid. Below the phase transition, the polarization approaches the theoretical maximum for total immobilization while above the phase transition the fluorescence becomes nearly completely depolarized. The discontinuity in the temperature dependence of polarization occurs within a temperature range under 5 degrees C in the case of pure phospholipids, but for mixed phospholipids occurs over a temperature range greater than 20 degrees C. From these data, phase diagrams describing the gel-sol equilibrium can be constructed; the phase diagrams correspond well with those described in the literature which were constructed using spin-label probes or from x-ray diffraction patterns. The marked change in polarization at the phase transition may be related to the packing of the probe molecule into the lipid bilayer: fluorescence measurements on oriented bilayers indicate that below the phase transition the long axis of the probe is oriented perpendicular to the plane of the membrane while above the transition the probe is oriented randomly relative to the plane of the membrane.

Sarcoplasmic reticulum: VIII. Use of 8-anilino-1-naphthalene sulfonate as conformational probe on biological membranes

Abstract The fluorescence of 8-anilino-1-naphthalene sulfonate (ANS) 3 is enhanced by skeletal muscle microsomes and micellar dispersions of phospholipids. The magnitude of the enhancement is a unique function of the ionic composition, rising with the concentration of cations according to a titration curve. The effect of cations might reflect the increased hydrophobic character of the membrane or the increased binding of ANS, induced by cations. The optimum pH for the ANS fluorescence in the presence of microsomes or lecithin is at pH 1–4 and at pH 7.3 a complex temperature dependence was observed with indications of a transition at 35–40 °. Treatment of microsomes or lecithin with phospholipase C causes a decrease of ANS fluorescence while trypsin digestion had little or no effect. Polymyxin B, a circular polypeptide, inhibits the ATPase activity and Ca 2+ transport of muscle microsomes accompanied by a large increase in fluorescence enhancement in the presence of ANS. Tyrocidine also inhibited the biochemical functions while gramicidin was less effective. Polyene antibiotics were generally without effect. These observations indicate that caution is required in the evalution of ANS fluorescence data in terms of conformational changes in membrane proteins alone, since the contribution of phospholipids to the fluorescence is significant.

X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers

Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

Water structure changes induced by hydrophobic and polar solutes revealed by simulations and infrared spectroscopy

A combination of simulations and Fourier transform infrared spectroscopy was used to examine the effect of three ionic solutes (KCl, NaCl, and KSCN), the polar solute urea, and the osmolyte trimethylamine-N-oxide (TMAO) on a water structure. The ionic solutes increase the mean water–water H-bond angle in their first hydration shell concomitantly shifting the OH stretching mode to higher frequency, and shifting the HOH bending mode to lower frequency. TMAO decreases the mean water–water H-bond angle in its first hydration shell, shifts the OH stretching mode frequency down, and shifting the HOH bending mode frequency up. Urea has no effect on the mean H-bond angle, OH stretch, and HOH bend frequencies. These results can be explained in terms of changes in the relative proportions of two H-bond angle populations: Ionic solutes increase the population of more distorted (larger angle) H bonds relative to the less distorted population, TMAO has the reverse effect, while urea does not affect the H-bond angle probability distribution. The negligible effect of urea on water structure supports the direct binding model for urea-induced protein denaturation.A combination of simulations and Fourier transform infrared spectroscopy was used to examine the effect of three ionic solutes (KCl, NaCl, and KSCN), the polar solute urea, and the osmolyte trimethylamine-N-oxide (TMAO) on a water structure. The ionic solutes increase the mean water–water H-bond angle in their first hydration shell concomitantly shifting the OH stretching mode to higher frequency, and shifting the HOH bending mode to lower frequency. TMAO decreases the mean water–water H-bond angle in its first hydration shell, shifts the OH stretching mode frequency down, and shifting the HOH bending mode frequency up. Urea has no effect on the mean H-bond angle, OH stretch, and HOH bend frequencies. These results can be explained in terms of changes in the relative proportions of two H-bond angle populations: Ionic solutes increase the population of more distorted (larger angle) H bonds relative to the less distorted population, TMAO has the reverse effect, while urea does not affect the H-bond angle pr...

Interaction of general anesthetics with phospholipid vesicles and biological membranes

Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether and chloroform are observed to shift the phase transitions of phospholipid vesicles to lower temperatures, and from these data partition coefficients for the anesthetic between lipid and water can be calculated. In contrast to the anesthetics, high concentrations of ethanol are required to shift the phase transition of lipids and glycerol causes no effect. Above the phase transition general anesthetics alter nuclear magnetic resonance spectra of phospholipid dispersions and increase the rotational and lateral diffusion rates of fluorescent probes located in the hydrocarbon core of the bilayer, indicating that they induce disorder in the structure. In red blood cell membranes and sarcoplasmic reticulum fragments, the rotational diffusion rate of 1-phenyl-6-phenylhexatriene is increased in the presence of general anesthetics. The 220 MHz nuclear magnetic resonance spectra of sarcoplasmic reticulum reveal some resolved lines from the lecithin fatty acid protons; addition of general anesthetic increases the contribution of these peaks. The data from the NMR and fluorescence techniques lead to the conclusion that general anesthetics increase the pool size of melted lipids in the bimolecular phospholipid layers of biological membranes; this would account for the ability of general anesthetics to increase passive diffusion rates of various substances in membranes.

The hydration of amides in helices; a comprehensive picture from molecular dynamics, IR, and NMR

We examined the hydration of amides of α3D, a simple, designed three‐helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy (∼50%–70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I′ band) of a group of surface Ala residues. The surface‐exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I′ bands of the buried 13C‐labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen‐deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4–7 residues N‐terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 310/α/π‐helical conformation.

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Abstract Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca 2+ , Mg 2+ , and Mn 2+ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current. On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca 2+ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca 2+ , Mg 2+ , or Mn 2+ are detected. A23187 produces fluorescent transients under conditions of Ca 2+ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca 2+ content. Changes in A23187 fluorescence due to mitochondrial Ca 2+ accumulation are much smaller by comparison and fluorescence transients are not detected. Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca 2+ and Mg 2+ , and less than 2 nsec in the presence of excess EDTA. The present results are consistent with a model of ionophore-mediated cation transport in which free M 2+ binds with A23187 at the membrane surface to form the complex M(A23187) + . Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187) 2 , which carries the divalent cation through the membrane. These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.

Cytochrome c interaction with membranes: I. Use of a fluorescent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes☆

Quenching of 12-(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS3 fluorescence is biphasic (t12 below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids. Addition of cytochrome c to cytochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytochrome c-depleted mitochondria is 1.8 × 106 m, while the affinity constant for reduced cytochrome c is 0.5 × 106 m. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance with midpoint potential differences between the bound and free forms.

Tryptophan phosphorescence at room temperature as a tool to study protein structure and dynamics.

Fluorescence and phosphorescence resemble each other and in many ways can give the same type of information. Both originate from a dipolar interaction between light and the molecule. In this regard, both are polarized and subject to the same type of quenching phenomena. In other respects the information which they divulge are complementary. The fluorescence quantum yield is higher for exposed tryptophans and this is expressed in longer lifetime (Grinvald and Steinberg, 1976); in contrast long lifetime of phosphorescence appears to correlate with burial. Phosphorescence, spin-disallowed, is much longer lived than fluorescence. This allows the structural/dynamic characterization of proteins to be studied on a new time regime. A really remarkable finding of studies of protein phosphorescence is that there is such variability both in phosphorescence lifetime and quenchability. We would interpret this to indicate that the tryptophan environment can range from essentially a crystal, almost comparable in rigidity as found at 77 K, to tryptophans in a flexible environment, almost as flexible as free in solution. An interesting task will be to examine the relationship between the yield and lifetime of phosphorescence and details of the tryptophan environment in terms of rigidity and adjacent amino acids among the proteins with known three dimensional structure.

A new method for measuring oxygen concentration in biological systems.

The oxygen dependent quenching of phosphorescence, if measured by the oxygen dependence of the phosphorescence lifetime, can be used as a sensitive and precise measure of oxygen concentration. The result is an optical method for measuring oxygen which is easily calibrated and the calibration may, depending on the experimental conditions, be stable for months to years. Although much remains to be done to realize the full potential of this method of measuring oxygen, it has great promise and we are proceeding with its development.