Incorporation of highly purified melittin into phosphatidylcholine bilayer vesicles

Melittin free of phospholipase A2 was prepared. In the absence of salt this highly pure protein starts to aggregate in solution at a protein concentration of Cp greater than 10(-3) M. In high salt solution (2 M) aggregation starts at Cp greater than 10(-6) M. This was determined from the blue shift of the intrinsic fluorescence of the protein. Reinvestigation of the quenching behaviour clearly shows that self-aggregation cannot be deduced from quenching experiments using nitrate or 2,2,6,6-tetramethylpiperidine-1-oxyl as quencher. The incorporation of melittin into phosphatidylcholine bilayer vesicles was studied by fluorescence quenching and by energy-transfer experiments using 2- and 6-anthroyloxypalmitic acid as acceptor and peptide tryptophan as donor. Incorporation of melittin into small unilamellar vesicles was found to be reduced below the lipid phase transition temperature, Tt, whereas it incorporates and distributes more randomly above Tt. Cooling the temperature below Tt after incubation at T greater than Tt leads to a deeper incorporation of the peptide into the lipid bilayer due to electrostatic interaction between the lipid phosphate groups and the positively charged amino acids. This stabilizing effect is lost above Tt and melittin is extruded to the polar phase. Quenching experiments support this finding. EPR measurements clearly demonstrate that even in the presence of high amounts of melittin up to 10 mol% with respect to the lipid broadening of the phase transition curves was only observed with fatty acid spin labels, where the doxyl group is localized near the bilayer surface. The order degree of the inner part of the bilayer remains almost unchanged even in the presence of high melittin content.     

Effect of micelle diameter on tryptophan dynamics in an amphipathic helical peptide in phosphatidylcholine

The effect of dimyristoylphosphatidylcholine (DMPC) on the conformation and environment of the single tryptophan residue of a model amphipathic helical polypeptide has been investigated by fluorescence quenching with a water-soluble, neutral quencher (acrylamide) and multiple-frequency phase fluorometry. The peptide H-Ser-Ser-Ala-Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Ly s-Glu- Ala-Phe-Ser-Ser-Ser-OH [18As; Kanellis, P., Romans, A.Y., Johnson, B.J., Kercret, H., Chiovetti, R., Jr., Allen, T.M., & Segrest, S.P. (1980) J. Biol. Chem. 255, 11464] was synthesized by solid-phase techniques. Peptide was incubated at 26 degrees C with DMPC at various peptide:lipid weight ratios. The diameter of the resulting disk-shaped micelles increases with increasing lipid concentration from 12.0 +/- 0.4 nm at a 1:1 weight ratio of peptide to lipid to a maximum of 48.7 +/- 1.0 nm at a 1:13 ratio. At a weight ratio of 1:5, the average diameter is 22.7 +/- 0.6 nm. Decreasing the peptide:lipid ratio of the micelle resulted in a blue-shift in the fluorescence emission maximum (from 337 nm at 1:1 to 334 nm at 1:5), an increase in the fluorescence lifetime of the tryptophan measured by the phase shift method at 18 MHz (from 3.12 ns at 1:1 to 3.61 ns at 1:5), a decrease in the rate of fluorescence quenching by acrylamide (from 0.87 x 10(9) M-1 s-1 at 1:1 to 0.42 x 10(9) M-1 s-1 at 1:5), and an increase in the activation energy for quenching (from 6.7 kcal/mol at 1:1 to 12.7 kcal/mol at 1:5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural-dynamic properties of the tryptophan residue environment in melittin

The structural dynamics of the environment of the single tryptophan residue in melittin was studied by site-selective red-edge-excitation fluorescence spectroscopy. The dependence of the spectral shift on transition from excitation in a maximum (at 280 nm) to long-wavelength edge (305 nm) was studied as a function of temperature. It was shown, that for melittin at high ionic strength (tetramer), the three regions of temperature dependence of the red-edge effect are observed: retarded relaxation (up to +30 degrees C), relaxational changes of spectra (from +30 to +50 degrees C) and thermal changes of structure (above +50 degrees C). The dipolar-re-orientational relaxation time and activation energy of orientation motions in the environment of indolic ring in the tetrameric melittin structure were estimated. Extrapolation from relaxational region to room temperature results in relaxation time 40 ns. In monomeric melittin (at low ionic strength) the red-edge shift of spectra is absent. The distinct differences in character of thermal quenching of fluorescence between monomeric and tetrameric forms of melittin are observed. It follows, that the short-wave-length fluorescence shift on monomer-tetramer transition is due to both the reduction of polarity, and the increase in rigidity of tryptophan environment, the absence of relaxation motions at nanosecond times.     

Fluorescence quenching studies of apolipoprotein A-I in solution and in lipid-protein complexes: protein dynamics

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 X 10(9) and 0.38 X 10(9) M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic alpha-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic alpha-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

The component analysis of tryptophan fluorescence spectra of melittin during its oligomerization

The oligomerization of melittin with increasing ionic strength and protein concentration was investigated using the methods of decomposition of its tryptophan fluorescence spectra into "elementary" log-normal components. At high ionic strength (up to 2 M KCl), the emission spectra of tetrameric melittin are well described as the sum of two log-normal components, suggesting the presence of tryptophan residues in two sorts of environment with greatly differing polarity. Measurements of fluorescence spectra by iodide showed that these two spectral components possess different Stern-Volmer constants, that is, the tryptophans emitting them have different solvent accessibility, which does not correlate with the crystallographic structure of tetrameric melittin. Moreover, in the oligomerization transition induced by ionic strength, the tetrameric intermediate is formed, which has log-normal spectral components with relative contributions differing from those in 2 M KCl.     

Amphipathic alpha-helix bundle organization of lipid-free chicken apolipoprotein A-I

Apolipoprotein A-I (apoA-I), the major protein component of plasma high-density lipoprotein (HDL), exists in alternate lipid-free and lipid-bound states. Among various species, chicken apoA-I possesses unique structural properties: it is a monomer in the lipid-free state and it is virtually the sole protein component of HDL. Near-UV circular dichroism (CD) spectroscopic studies provide evidence that chicken apoA-I undergoes a major conformational change upon binding to lipid, while far-UV CD data indicate its overall alpha-helix content is maintained during this transition. The fluorescence emission wavelength maximum (excitation 295 nm) of the tryptophans in apoA-I (W74 and W107) displayed a marked blue shift in both the lipid-free (331 nm) and HDL-bound (329 nm) states, compared to free tryptophan in solution. The effect of aqueous quenchers on tryptophan fluorescence was determined in lipid-free, dimyristoylphosphatidylcholine (DMPC)- and HDL-bound states. The most effective quencher in the lipid-free and HDL-bound states was acrylamide, giving rise to Ksv values of 1.6 +/- 0.1 and 1.2 +/- 0.1 M-1, respectively. Together, these data suggest that a hydrophobic environment around the two tryptophan residues (W74 and W107) is maintained in alternate conformations of the protein. To further probe the molecular organization of lipid-free apoA-I, its effect on the fluorescence properties of 8-anilino-1-naphthalenesulfonic acid (ANS) was determined. Human and chicken apoA-I induced a similar increase in ANS fluorescence quantum yield, in keeping with the hypothesis that these proteins adopt a similar global fold in the absence of lipid. When considered with near- and far-UV CD experiments, the data support a model in which lipid-free chicken apoA-I is organized as an amphipathic alpha-helix bundle. In other studies, lipid-soluble quenchers, 5-, 7-, 10-, and 12-DOXYL stearic acid (DSA), were employed to investigate the depth of penetration of apoA-I into the surface monolayer of spherical HDL particles. 5-DSA was the most effective quencher, suggesting that apoA-I tryptophan residues localize near the surface monolayer, providing a structural rationale for the reversibility of apoA-I-lipoprotein particle interactions.     

Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.

Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.     

Melittin-GM1 interaction: a model for a side-by-side complex

We report here the interaction of melittin with ganglioside GM1 by steady-state fluorescence, one-dimensional (1)H NMR spectroscopy and molecular modeling. In the presence of GM1 the emission maximum of melittin is blue shifted and fluorescence quenching efficiencies of iodide and acrylamide are substantially reduced, indicating a shielding of tryptophan of melittin from aqueous environment. Significant line broadening of NMR resonances of melittin, suggestive of motional restriction, is observed. Molecular modeling indicates a melittin-GM1 complex with N-terminal hydrophobic stretch of melittin associating with the ceramide tail and C-terminal hydrophilic end of melittin having favorable electrostatic interaction with the carbohydrate head group of GM1.     

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.     

The principles of analysis on multicomponent fluorescence spectra of proteins. Temperature and pH-induced transitions in tetrameric melittin

Structural transitions in the tetrameric melittin from bee venom in 2 M KCl induced by variations of pH (from 0.7 to 12.0) and temperature (from 2 to 95 degrees C) have been studied. The pH and temperature ranges of structural changes and the zones of emission quenching of discerning tryptophan classes were revealed. The analysis of the temperature dependence of monotonic changes of spectral maximum positions and relative fluorescence yields allowed one to discriminate the zone of structural transitions in the tetramer from that of temperature quenching due to the thermal activation of fluorophore collisions with neighboring quenching groups in protein. Based on the new and earlier published results, some advantages and modes of using the method of component analysis of protein tryptophan spectra were summarized to determine the main characteristics of physicochemical transitions in proteins.     

Static quenching of tryptophan fluorescence by oxidized dithiothreitol

The quenching of fluorescence of 5-methoxyindole, N-acetyl-L-tryptophanamide and two single tryptophan containing peptides, melittin and mastoparan X, by oxidized dithiothreitol was studied. The slopes of the Stern-Volmer plots for steady-state fluorescence quenching were 133 M-1, 71.2 M-1, 75.5 M-1 and 35.0 M-1 at 21 degrees C and pH 7.0 for 5-methyoxyindole, N-acetyl-L-tryptophanamide, melittin and mastoparan X respectively. Fluorescence lifetimes of indole or tryptophan in these compounds, as determined by multifrequency phase fluorometry, were decreased by 15% or less at concentrations that produced 50% or more quenching of steady-state fluorescence. Thus, quenching of fluorescence by oxidized dithiothreitol for these derivatives of indole appears to be largely static in nature, suggesting a ground-state interaction.     

Spectrofluorimetric studies on C-terminal 34 kDa fragment of caldesmon

Analysis of the tryptophan fluorescence emission spectra of caldesmon and its 34 kDa C-terminal fragment indicates that all tryptophan residues are located on the surface of the molecule, accessible to solvent. All three tryptophan residues of the 34 kDa fragment and four of the five tryptophan residues of intact protein are accessible to free water, whereas one located in the N-terminal region of molecule is accessible only to bound water molecules. The temperature dependence of the fluorescence parameters indicates higher thermal stability of the 34 kDa fragment than the whole caldesmon molecule. The interaction of the 34 kDa fragment of caldesmon (like that of the intact molecule) with calmodulin is accompanied by a blue shift of the fluorescence emission maximum and an increase in the relative quantum yield. Computer-calculated binding constants show that the binding of calmodulin to the 34 kDa fragment (K = 2.5 x 10(5) M-1) is of two orders of magnitude weaker than that to intact caldesmon (K = 1.4 x 10(7) M-1). The interaction with tropomyosin results in a blue shift of the spectrum of the 34 kDa fragment, yet there is no effect on the spectrum of intact caldesmon. Binding constants of tropomyosin to caldesmon (K = 3.8 x 10(5) M-1) and its 34 kDa fragment (K = 2.3 x 10(5) M-1) are similar. Binding of calmodulin to caldesmon and to the 34 kDa fragment affects their interaction with tropomyosin.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid binding of the exchangeable apolipoprotein apolipophorin III induces major changes in fluorescence properties of tryptophans 115 and 130

The effect of lipid association on the local environment of the two tryptophan residues of Locusta migratoria apolipophorin III (apoLp-III) has been studied. In the lipid-free state, Trp115 in helix 4 is buried in the hydrophobic interior of the helix bundle, while Trp130 is located in a loop connecting helices 4 and 5. Fluorescence spectroscopy of single Trp mutants revealed an emission maximum (lambda(max)) of 321 nm for apoLp-III-W@115 (excitation 280 nm) which red-shifted to 327 nm upon binding to dimyristoylphosphatidylcholine (DMPC). ApoLp-III-W@130 displayed a lambda(max) of 338 nm while interaction with DMPC resulted in a blue shift to 331 nm. Quenching studies with KI and acrylamide revealed decreased accessibility to Trp115 compared to Trp130, while lipid binding induced a decrease in quenching of Trp130. Aromatic circular dichroism (CD) spectra showed that Trp vibronic transitions at 278, 286, and 294 nm for lipid-free apoLp-III were caused by Trp115. Upon lipid association, aromatic extrema are reversed in sign, becoming entirely negative with both Trp residues contributing to the vibronic transitions, implying restriction in side-chain mobility of these residues. Thus, lambda(max), quencher accessibility, and aromatic CD analysis indicate that Trp115 is much less solvent-exposed than Trp130. Differences in fluorescence properties of these residues are minimized in the lipid-bound state, a result of relocation of Trp115 and Trp130 into the lipid milieu. Thus, in addition to the hydrophobic faces of apoLp-III amphipathic alpha-helices, the loop region containing Trp130 comes in close contact with DMPC.     

Time-resolved fluorescence spectroscopy of human adenosine deaminase: effects of enzyme inhibitors on protein conformation

Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.     

Red-edge-excitation fluorescence spectroscopy of single-tryptophan proteins

With the aim of finding non-equilibrium dipole-relaxational electronic excited states of tryptophan residues in proteins the dependence of the fluorescence emission maximum on excitation wavelength was studied for several proteins containing a single tryptophan residue per molecule. Spectral shifts upon red-edge excitation are not observed for short wavelength-emitting proteins (azurin, two-calcium form of whiting parvalbumin, ribonucleases C ₂ and T ₁). This may be because of the non-polar environment of the tryptophan residues in these proteins or because of the absence of dipole-orientational broadening of spectra. The effect was also not found for proteins emitting at long wavelengths (max. at 341–350 nm) —melittin at low ionic strength, IT-Aj1 protease inhibitor, myelin basic protein. In these proteins, the tryptophan residues are exposed to the rapidly relaxing aqueous solvent. Spectral shifts associated with red-edge excitation are observed for proteins emitting in the medium spectral range — human serum albumin in the N and F forms, IT-Aj1 protease inhibitor at pH 2.9, melittin at high ionic strength as well as the albumin-dodecylsulfate complex. This suggests the existence in these proteins of a distribution of microstates for tryptophan environment with various orientation of dipoles and of slow (on the nanosecond time scale) mobility of the field of these dipoles. As a result the emission proceeds from electronic excited states which are not at equilibrium.     

pH-dependent quenching of the fluorescence of tryptophan residues in class A beta-lactamase from E. coli (TEM-1)

We performed an investigation of the pH-dependent quenching of the fluorescence of tryptophan residues of TEM-1 beta-lactamase from E. coli by uncharged and charged quenchers. pH-dependent Stern-Volmer constants (Ksv/pH) of tryptophan residues allowed us to determine subtle but discrete structurally and functionally important processes.     

Early conformational changes and activity modulation induced by guanidinium chloride on intestinal alkaline phosphatase.

Moderate concentrations of guanidinium chloride induce both instantaneous and time-dependent modifications of the catalytic and optical properties of intestinal alkaline phosphatase, which undergoes consecutive conformational transitions at about 0.05 M, 0.25 M and 1.0 M denaturant. A paradoxical activation is observed up to 1.0 M-guanidine, with a maximum at 0.25 M- and a mid-point around 0.5 M-guanidine. Difference absorbance and fluorescence spectra imply a change in the state of ionization of the protein residues, with variation in molecular size suggested by light-scattering. Random-coil formation is indicated by a lower fluorescence yield, a more polar environment of the aromatic residues and another separate tryptophan emission. Iodide quenching confirms the alterations of conformation. Deprotonation favours the loss of the intramolecular constraints and the enhancement of the structure disruption by guanidine.     

Motions studies of the human alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and polarization of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues.

Dynamics studies on tryptophan residues of human alpha 1-acid glycoprotein (orosomucoid) and of 2-p-toluidinylnaphthalene-6-sulfonate bound to the protein are performed. Excitation at the red edge of the absorption spectrum of the tryptophan does not lead to a shift of the fluorescence emission maximum of the fluorophore. This reveals that Trp residues present motions with respect to their microenvironment. This is confirmed by polarization studies as a function of temperature. Excitation at the red edge of the absorption spectrum of TNS leads to an important shift (15 nm) of the fluorescence emission maximum of the probe. This reveals that emission of TNS occurs before relaxation of the amino-acids dipole occurs. Emission from a non-relaxed state means that TNS molecules are bound tightly to the protein, a result confirmed by polarization studies.     

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 degrees C as compared to that at 5 degrees C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC complexes.