Anisotropy decays of indole, melittin monomer and melittin tetramer by frequency-domain fluorometry and multi-wavelength global analysis

We used frequency-domain fluorescence spectroscopy to measure the fluorescence lifetime and anisotropy decays of indole in propylene glycol, and of the tryptophan emission of melittin monomer and tetramer in water solutions at 5 degrees C. We obtained an increase in resolution of the anisotropy decays by using multiple excitation wavelengths, chosen to provide a range of fundamental anisotropy values. The multi-excitation wavelength anisotropy decays were analyzed globally to recover a single set of correlation times with wavelength-dependent anisotropy amplitudes. Simulated data and kappaR2 surfaces are shown to reveal the effect of multi-wavelength data on the resolution of complex anisotropy decays. For both indole and melittin, the anisotropy decays are heterogeneous and require two correlation times to fit the frequency-domain data. For indole in propylene glycol at 5 degrees C we recovered correlation times of 0.59 and 4.10 ns, which appear to be characteristic of the rigid and asymmetric indole molecule. For melittin monomer the correlation times were 0.13 and 1.75 ns, and for melittin tetramer 0.12 and 3.96 ns. The shorter and longer correlation times of melittin are due to segmental motions and overall rotational diffusion of the polypeptide.     

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.     

Resolution of complex anisotropy decays by variable frequency phase-modulation fluorometry: a stimulation study

We used simulations to determine the resolution of complex anisotropy decay laws which is obtainable by frequency-domain fluorometry. The simulations include the effects of torsional and segmental motions of tryptophan residues in proteins, the multiple correlation times of asymmetric molecules, and three-component anisotropy decays. For a protein with a global correlation time of 10 ns it should be possible to resolve torsional motions with correlation times as short as 10 ps if the amplitude of the rapid motion is at least 20% of the total anisotropy decay with r0 = 0.4. Correlation times which differ by only 1.4-fold can be resolved, making this method useful for determination of the shape of proteins and other asymmetric molecules. It is possible to resolve three-component anisotropy decays if the overall difference among the correlation times is 30-fold. Such resolution will be useful for understanding of internal motions of proteins and membranes. The validity of these predictions is demonstrated in the subsequent paper using experimental data for melittin in solution and when bound to membranes (Maliwal, B.P., Hermetter, A. and Lakowicz, J.R. (1986) Biochim. Biophys. Acta 873, 173-181).     

Picosecond resolution of tyrosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry

We extended the technique of frequency-domain fluorometry to an upper frequency limit of 2000 MHz. This was accomplished by using the harmonic content of a laser pulse train (3.76 MHz, 5 ps) from a synchronously pumped and cavity-dumped dye laser. We used a microchannel plate photomultiplier as the detector to obtain the 2-GHz bandwidth. This new instrument was used to examine tyrosine intensity and anisotropy decays from peptides and proteins. These initial data sets demonstrate that triply exponential tyrosine intensity decays are easily recoverable, even if the mean decay time is less than 1 ns. Importantly, the extended frequency range provides good resolution of rapid and/or multiexponential tyrosine anisotropy decays. Correlation times as short as 15 ps have been recovered for indole, with an uncertainty of +/- 3 ps. We recovered a doubly exponential anisotropy decay of oxytoxin (29 and 454 ps), which probably reflects torsional motions of the phenol ring and overall rotational diffusion, respectively. Also, a 40-ps component was found in the anisotropy decay of bovine pancreatic trypsin inhibitor, which may be due to rapid torsional motions of the tyrosine residues and/or energy transfer among these residues. The rapid component has an amplitude of 0.05, which is about 16% of the total anisotropy. The availability of 2-GHz frequency-domain data extends the measurable time scale for fluorescence to overlap with that of molecular dynamics calculations.     

A study of protein dynamics from anisotropy decays obtained by variable frequency phase-modulation fluorometry: internal motions of N-methylanthraniloyl melittin

Internal motions of melittin and its lipid complexes were studied by anisotropy decays determined by frequency-domain fluorometry. A covalent anthraniloyl probe was attached, probably to lysine-21. The emission spectra indicate that the anthraniloyl moiety is exposed to solvent in both monomeric and tetrameric forms and is present at the lipid-water interfacial region in the lipid complexes. The fluorescence intensity decay of melittin in solution and its lipid complexes was characterized by three lifetimes. The lifetimes were near 1-2 ns, 6-7 ns and 10 ns. At increased temperatures there was an increase in the amplitude of the intermediate lifetime and a decrease in that of the longer lifetime. For all the melittin systems, at least three correlation times were required to fit the anisotropy data. Of the three correlation times, the shortest correlation time represents the local motions of the probe, while the longest represents global motions of the whole molecule. The intermediate correlation time probably represents the dynamics of domains/helices within the molecule. The melittin monomer is highly flexible, with greater than 90% of its anisotropy being lost by the local motions. Even though it is well organized (greater than 75% helical), the tetramer is still a highly flexible molecule, with 70% of its anisotropy being lost by the local motions. The internal motions of melittin decrease upon binding to lipids and are sensitive to the phase state of the lipid complexes.     

Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry

Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy.     

Frequency-domain measurements of the rotational dynamics of the tyrosine groups of calmodulin

We used frequency-domain fluorometry to determine the intensity and anisotropy decay kinetics of tyrosine residues in calmodulin and its fragments. Excitation was provided by a continuous ultraviolet laser source, a frequency-doubled rhodamine 6G ring dye laser, whose output was externally modulated to 200 MHz. Both the intensity and anisotropy decays were found to be multiexponential and dependent upon temperature and solution conditions. By examination of calmodulin fragments we determined that energy transfer between the two tyrosine residues reduces the steady-state anisotropy values by about 20%. Additionally, the frequency-domain anisotropy decays indicate local torsional motions of the tyrosine residues, as well as significant individual motions of the two domains of calmodulin.     

Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry

We used 2 GHz harmonic content frequency-domain fluorescence to measure the intensity and the anisotropy decays from the intrinsic tryptophan fluorescence from human hemoglobin (Hb). The tryptophan intensity decays are dominated by a short-lived component which accounts for 35-60% of the total steady state intensity. The decay time of this short component varies from 9 to 27 ps and this component is sensitive to the ligation state of Hb. Our error analyses indicate the uncertainty is about +/- 3 ps. The intensity decays also show two longer lived components near 0.7 and 8 ns, which are probably due either to impurities or to Hb molecules in conformations which do not permit energy transfer. The anisotropy decays indicate the tryptophan residues in Hb are highly mobile, with apparent correlation times near 55 ps.     

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T ₁, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40°C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T ₁, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.Abbreviations Ac acrylamide - ACTH adrenocorticotropin hormone (1–24) - BPTI bovine pancreatic trypsin inhibitor - NATA N-acetyl-L-tryptophanamide - RNase T ₁ ribonuclease T ₁ - S. Nuclease staphylococcus aureus nuclease Supported by grants DMB-8804931 and DIR-8710401 from the National Science Foundation, and GM-39617 from the National Institutes of Health. J. R. Lakowicz acknowledges support from the Medical Biotechnology Center at the University of Maryland. I. Gryczynski was on leave from University of Gáansk, Institute of Experimental Physics, Gdansk, Poland, with partial support from CPBP 01.06.2.01 (Poland). H. Cherek was on leave from Nicholas Copernicus University, Torun, Poland, with partial support from CPBP 01.06.2.03 Offprint requests to: J. R. Lakowicz     

Anisotropy decay of fluorescence as an experimental approach to protein dynamics

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.     

Construction and performance of a variable-frequency phase-modulation fluorometer

We describe the construction and performance of a variable-frequency phase-modulation fluorometer. This instrument, which provides modulation frequencies from 1 to 200 MHz, was constructed using commercially available components. To facilitate the introduction of these instruments into other laboratories we describe in detail the chosen components and the principles of operation. The present light source is a continuous-wave helium-cadmium laser, which provides convenient excitation wavelengths of 325 and 442 nm. Modulation of the incident light is provided by one of several electro-optic modulators. The extent of modulation ranges from 1.0 to 0.2 as the frequency increases from 1 to 200 MHz. Phase angles and demodulation factors are measured using the cross-correlation method. The closely spaced frequencies are provided by two direct frequency synthesizers. The phase and modulation measurements are accurate to 0.2 degrees and 0.002, respectively, from 1 to 200 MHz. This accuracy allows considerable resolution of complex decay laws. The usefulness of frequency-domain fluorometry for the resolution of multiexponential decays is illustrated by the analysis of several difficult mixtures. As examples, we resolved a two-component mixture of anthracene (4.1 ns) and 9,10-diphenylanthracene (6.3 ns), and confirmed that the intensity decay of NADH in aqueous buffer is at least a double exponential (0.2 and 0.86 ns). We also resolved an especially difficult mixture of anthracene (4.1 ns) and 9-methylanthracene (4.5 ns), and a three-component mixture with decay times of 1.3, 4.1 and 7.7 ns. Frequency-domain fluorometers appear to be particularly useful for determination of complex decays of fluorescence anisotropy. This capability is illustrated by the determination of rotational correlation times as short as 47 ps for p-bis[2-(5-phenyloxazolyl)]benzene (POPOP) in hexane at 40 degrees C, and by the resolution of the two correlation times of anisotropic rotators such as perylene and 9-aminoacridine. Resolution of two anisotropy decay times for 9-aminoacridine is a difficult test because these correlation times differ by less than 2-fold. The resolution of multiexponential decays of intensity and anisotropy possible with this instrument is at least equivalent to that obtained using state-of-the-art time-resolved instruments based on mode-locked laser sources. The ease and rapidity of frequency-domain measurements, the relative simplicity of the equipment, the accuracy of the measurements and the lack of significant systematic errors indicate that frequency-domain fluorometry will be widely useful in chemical and biochemical research.     

Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.     

Studies of the fluorescence from tryptophan in melittin

The fluorescence lifetime and rotational correlation time of the tryptophan residue in melittin, as both a monomer and tetramer, have been measured between pH 6 and 11. The fluorescence decays are non-exponential and give lifetimes of 0.7±0.1 ns and 3.1±0.1 ns. This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. In a dilute solution of monomer the mean fluorescence lifetime is 2.3±0.1 ns, below pH 10, but falls to 1.7 ns at higher pH. In contrast, the melittin tetramer has a mean fluorescence lifetime of only 2.2 ns at pH 6, which falls to 1.9 ns by pH 8, and falls again above pH 10 to the same value as in monomeric melittin. The behaviour between pH 6 and 8 is explained as the quenching of the Trp residue by lysine groups, which are near to the Trp in the tetramer but in the monomer, are too distant to quench. Fluorescence anisotropy decays show that the Trp residue has considerable freedom of motion and the range of wobbling motion is 35±10° in the tetramer     

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.     

Anisotropy decay of diphenylhexatriene in melittin-phospholipid complexes by multifrequency phase-modulation fluorometry

Frequency-domain fluorometry was used to investigate the intensity and anisotropy decays of diphenylhexatriene (DPH) in melittin-lipid complexes. Simulated and experimental data indicate that correlation times ranging from 0.3 to 500 ns can be determined using data from 1 to 200 MHz. For the melittin-lipid complexes the hindered rotator model was not adequate to account for the anisotropy decays, especially at temperatures above the transition temperatures. At high protein-to-lipid ratios the data revealed the formation of small particles (100 A) of melittin and dipalmitoylphosphatidylcholine and the disruption of membrane order in bilayers of dipalmitoylphosphatidic acid.     

Effects of hydrophobic and hydrophilic bile salt mixtures on cholesterol crystallization in model biles

[Ru(2,2'-bipyridine)(2)(4,4'-dicarboxy-2,2'-bipyridine)](2+) (RuBDc) is a very photostable probe that possesses favorable photophysical properties including long lifetime, high quantum yield, large Stokes' shift, and highly polarized emission. In the present study, we demonstrated the usefulness of this probe for monitoring the rotational diffusion of high-molecular-weight (MW) proteins. Using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source, we compared the intensity and anisotropy decays of RuBDc conjugated to immunoglobulin G (IgG) and immunoglobulin M (IgM), which show a six-fold difference in MW We obtained slightly longer lifetimes for IgM (=428 ns in buffer) than IgG (=422 ns in buffer) in the absence and presence of glycerol, suggesting somewhat more efficient shielding of RuBDc from water in IgM than in IgG. The anisotropy decay data showed longer rotational correlation times for IgM (1623 and 65.7 ns in buffer) as compared to IgG (264 and 42.5 ns in buffer). Importantly, the ratio of the long rotational correlation times of IgM to IgG in buffer was 6.2, which is very close to that of MW of IgM to IgG (6.0). The shorter correlation times are most likely to be associated with domain motions within the proteins. The anisotropy decays reflect both the molecular size and shape of the immunoglobulins, as well as the viscosity. These results show that RuBDc can have numerous applications in studies of high-MW protein hydrodynamics and in fluorescence polarization immunoassays (FPI) of high-MW analytes.     

Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high repetition rate lasers, a phenomenon we call "light quenching." We now describe the theory of light quenching and some of its effects on the steady-state and time-resolved intensity and anisotropy decays of fluorophores. Light quenching can decrease or increase the steady-state or time-zero anisotropy. Remarkably, the light quenching can break the usual z axis symmetry of the excited-state population, and the emission polarization can range from -1 to +1 under selected conditions. The measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The effects of light quenching are different for a single pulse, which results in both excitation and quenching, as compared with a time-delayed quenching pulse. Time-delayed light quenching pulses can result in step-like changes in the time-dependent intensity or anisotropy and are predicted to cause oscillations in the frequency-domain intensity and anisotropy decays. The increasing availability of pulsed laser sources offers the opportunity for a new class of two-pulse or multiple-pulse experiments where the sample is prepared by an excitation pulse, the excited state population is modified by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.     

Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.