Fluorescence lifetime distributions in proteins.

The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most proteins can be satisfactorily described only using several exponential components. Here it is proposed that continuous lifetime distributions can better represent the observed decay. An approach based on protein dynamics is presented, which provides fluorescence lifetime distribution functions for single tryptophan residue proteins. First, lifetime distributions for proteins interconverting between two conformations, each characterized by a different lifetime value, are derived. The evolution of the lifetime values as a function of the interconversion rate is studied. In this case lifetime distributions can be obtained from a distribution of rates of interconversion between the two conformations. Second, the existence of a continuum of energy substates within a given conformation was considered. The occupation of a particular energy substate at a given temperature is proportional to the Boltzmann factor. The density of energy states of the potential well depends upon the width of the well, which determines the degree of freedom the residue can move in the conformational space. Lifetime distributions can be obtained by association of each energy substate with a different lifetime value and assuming that the average conformation can change as the energy of the substate is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational dynamics of unfolded peptides as a function of chain length: a frequency domain fluorescence approach

The fluorescence emission decays of single-tryptophan-containing peptides of different chain lengths in their unfolded state were investigated in the frequency domain. The data were analyzed using different functions, i.e., exponential fit and probability-density functions of different shape. We found that unimodal Lorentzian distributions best describe the fluorescence decays. This finding agrees with the point of view, now broadly accepted, that rapid motions exist in polypeptides. As a consequence of this flexibility, a large variety of conformations, with an unequal perturbation of tryptophan in its excited state, is generated. The lifetime distribution center was independent of the length of the polypeptide chain but strongly related to the nature of the amino acid residues located in the proximity of the tryptophan in the primary structure. The full width at half maximum, W, of the lifetime distribution was found to be related to the length of unfolded polypeptide by the empirical logarithmic relationship W = 0.83 log n, where n indicates the number of residues. For short peptides, a single lifetime or a narrow range of lifetimes is observed because of the fast relaxation of the tryptophanyl environment. On peptide lengthening, the spectrum of conformations, which the peptide can assume, increases; this causes a complex fluorescence decay represented by a lifetime distribution. For long polypeptide chains, the motions of the regions far from tryptophan do not significantly perturb the chromophore environment.     

Time-resolved fluorescence of the single tryptophan residue in rat alpha-fetoprotein and rat serum albumin: analysis by the maximum-entropy method.

The time-resolved fluorescence emissions of the lone tryptophan residues in rat alpha-fetoprotein (RFP) and rat serum albumin (RSA) were studied. The total fluorescence intensity decays in both proteins were multiexponential. Analysis of the data by nonlinear least squares as a sum of discrete exponentials showed that four exponentials were needed for a satisfactory fit for both proteins. Analysis by the maximum entropy method using 150 logarithmically equally spaced exponentials yielded four well-resolved excited-state lifetime classes with barycenters and relative amplitudes values (ci) that corresponded to those obtained from the nonlinear least-squares method. Changing the temperature affected the relative amplitudes of the lifetime classes but had little effect on the lifetime values themselves. This suggests that the four classes reflect local conformational substates that exchange slowly with respect to the time window of observation defined by the longest lifetime. The internal rotational dynamics of the tryptophan in each protein was monitored by fluorescence anisotropy decay measurements. The mobility of the tryptophan appeared to be larger and faster in RFP than in RSA. The nonlinear least-squares analysis suggests the existence of three rotational correlation times of 0.1, 3, and 55 ns for this protein. As a function of temperature, the long correlation time did not follow the Perrin's law expected for a rigid rotating body. This suggests that this correlation time may reflect not only the Brownian rotation of the whole protein but also the flexibilities of domains in the protein. For RSA a two-component model with correlation times of 0.4 and 31 ns was sufficient to describe the data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolvability of fluorescence lifetime distributions using phase fluorometry.

The analysis of the fluorescence decay using discrete exponential components assumes that a small number of species is present. In the absence of a definite kinetic model or when a large number of species is present, the exponential analysis underestimates the uncertainty of the recovered lifetime values. A different approach to determine the lifetime of a population of molecules is the use of probability density functions and lifetime distributions. Fluorescence decay data from continuous distributions of exponentially decaying components were generated. Different magnitudes of error were added to the data to simulate experimental conditions. The resolvability of the distributional model was studied by fitting the simulated data to one and two exponentials. The maximum width of symmetric distributions (uniform, gaussian, and lorentzian), which cannot be distinguished from single and double exponential fits for statistical errors of 1 and 0.1%, were determined. The width limits are determined by the statistical error of the data. It is also shown that, in the frequency domain, the discrete exponential analysis does not uniformly weights all the components of a distribution. This systematic error is less important when probability and distribution functions are used to recover the decay. Finally, it is shown that real lifetime distributions can be proved using multimodal probability density functions. In the companion paper that follows we propose a physical approach, which provides lifetime distribution functions for the tryptophan decay in proteins. In the third companion paper (Alcala, J.R., E. Gratton, and F.J. Prendergast, 1987, Biophys. J., in press) we use the distribution functions obtained to fit data from the fluorescence decay of single tryptophan proteins.     

Effects of temperature on the fluorescence intensity and anisotropy decays of staphylococcal nuclease and the less stable nuclease-conA-SG28 mutant

Frequency-domain fluorescence spectroscopy was used to investigate the effects of temperature on the intensity and anisotropy decays of the single tryptophan residues of Staphylococcal nuclease A and its nuclease-conA-SG28 mutant. This mutant has the beta-turn forming hexapeptide, Ser-Gly-Asn-Gly-Ser-Pro, substituted for the pentapeptide Tyr-Lys-Gly-Gln-Pro at positions 27-31. The intensity decays were analyzed in terms of a sum of exponentials and with Lorentzian distributions of decay times. The anisotropy decays were analyzed in terms of a sum of exponentials. Both the intensity and anisotropy decay parameters strongly depend on temperature near the thermal transitions of the proteins. Significant differences in the temperature stability of Staphylococcal nuclease and the mutant exist; these proteins show characteristic thermal transition temperatures (Tm) of 51 and 30 degrees C, respectively, at pH 7. The temperature dependence of the intensity decay data are shown to be consistent with a two-state unfolding model. For both proteins, the longer rotational correlation time, due to overall rotational diffusion, decreases dramatically at the transition temperature, and the amplitude of the shorter correlation time increases, indicating increased segmental motions of the single tryptophan residue. The mutant protein appears to have a slightly larger overall rotational correlation time and to show slightly more segmental motion of its Trp than is the case for the wild-type protein.     

Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects

Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.     

Fluorescence lifetime distributions in human superoxide dismutase. Effect of temperature and denaturation.

The internal dynamics of human superoxide dismutase has been studied using time-resolved fluorescence. The fluorescence decay has been analyzed using continuous distribution of lifetime values. The effect of temperature and conformational state on the lifetime distribution has been investigated. The emission of the single tryptophan residue depends on the nature and dynamics of the protein matrix. Conformational changes have been induced by increased concentration of guanidinium hydrochloride. We found that both temperature and conformation strongly effect the width of the lifetime distribution.     

Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study.

The unfolding of holo and apo forms of human Cu/Zn superoxide dismutase by guanidine hydrochloride has been investigated by steady-state and dynamic fluorescence. In agreement with previous observations, a stabilizing effect of the metal ions on the protein tertiary structure was apparent from comparison of apo- and holoproteins, which both showed a sharp sigmoidal transition though at different denaturant concentrations. The transition was also followed by circular dichroism to check the extent of secondary structure present at each denaturant concentration. The results are incompatible with a simple two-state mechanism for denaturation. The occurrence of a more complicated process is supported by the emission decay properties of the single tryptophanyl residue at different denaturant concentrations. A complex decay function, namely, two discrete exponentials or a continuous distribution of lifetimes, was always required to fit the data. In particular, the width of the lifetime distribution, which is maximum at the transition midpoint, reflects heterogeneity of the tryptophan microenvironment and thus the presence of different species along the denaturation pathway. In the unfolded state, the width of the lifetime distribution is broader than in the folded state probably because the tryptophan residue is affected by a larger number of local conformations. The dissociation of the dimer was also studied by varying the protein concentration at different denaturant concentrations. This process affects primarly the surface of the protein rather than its secondary structure as shown by a comparison between the tryptophan emission decay and circular dichroism data under the same conditions. Another consequence of dissociation is a greater instability in the structure of the monomers, which are more easily unfolded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved fluorescence of apoferritin and its subunits

The decay of the intrinsic fluorescence of the apoferritin polymer and its subunits has been studied by pulse and phase shift techniques. Both techniques show that the fluorescence decay of all the samples tested cannot be described by a single exponential function. The fluorescence decay data of the apoferritin subunits obtained with either technique can be fitted satisfactorily with a function resulting from the sum of two exponential components. However, the polymer data obtained with the high resolution phase shift technique operated either by synchrotron radiation or by a mode-locked argon ion laser can be fitted better using a bimodal gaussian continuous distribution of lifetime components. The molecular basis for this distribution of lifetime values may lie in the heterogeneity of the tryptophan environment generated by the assembly of the subunits into the polymer. The binding of the first 100 irons to apoferritin quenches the intrinsic fluorescence without affecting the lifetimes in a proportional way. This finding may be taken as an indication that the quenching of the tryptophan fluorescence induced by the binding of iron has both static and dynamic components.     

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles

The fluorescence emission properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dipalmitoyl-3-sn-phosphatidylcholine and 1,2-dimyristoyl-3-sn-phosphatidylcholine multilamellar vesicles have been measured by using multifrequency phase fluorometry. The fluorescence decay of DPH in the phospholipid vesicles has been analyzed by assuming either that the decay is made up of a discrete sum of exponential components or that the decay is made up of one or more continuous distributions of lifetime components. The fit of the decay curve using exponentials required at least two terms, and the reduced X2 was relatively large. The fit using a continuous distribution of lifetime values used two continuous components. Several symmetric distribution functions were used: uniform, Gaussian, and Lorentzian. The distribution function that best described the decay was the Lorentzian. The full width at half-maximum of the Lorentzian distribution was about 0.6 ns at temperatures below the phase transition temperature. At the phospholipid phase transition and at higher temperatures, the distribution became quite narrow, with a width of about 0.1 ns. It is proposed that the lifetime distribution is generated by a continuum of different environments of the DPH molecule characterized by different dielectric constants. Below the transition temperature in the gel phase, the dielectric constant gradient along the membrane normal determines the distribution of decay rates. Above the transition, in the liquid-crystalline phase, the translational and rotational mobility of the DPH molecule increases, and the DPH experiences an average environment during the excited-state lifetime. Consequently, the distribution becomes narrower.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fluorescence decay of tryptophan residues in native and denatured proteins.

The fluorescence decay kinetics at different ranges of the emission spectrum is reported for 17 proteins. Out of eight proteins containing a single tryptophan residue per molecule, seven proteins display multiexponential decay kinetics, suggesting that variability in protein structure may exist for most proteins. Tryptophan residues whose fluorescence spectrum is red shifted may have lifetimes longer than 7 ns. Such long lifetimes have not been detected in any of the denatured proteins studied, indicating that in native proteins the tryptophans having a red-shifted spectrum are affected by the tertiary structure of the protein. The fluorescence decay kinetics of ten denatured proteins studied obey multiexponential decay functions. It is therefore concluded that the tryptophan residues in denatured proteins can be grouped in two classes. The first characterized by a relatively long lifetime of about 4 ns and the second has a short lifetime of about 1.5 ns. The emission spectrum of the group which is characterized by the longer lifetime is red shifted relative to the emission spectrum of the group characterized by the shorter lifetime. A comparison of the decay data with the quantum yield of the proteins raises the possibility that a subgroup of the tryptophan residues is fully quenched. It is noteworthy that despite this heterogeneity in the environment of tryptophan residues in each denatured protein, almost the same decay kinetics has been obtained for all the denatured proteins studied in spite of the vastly different primary structures. It is therefore concluded that each tryptophan residue interacts in a more-or-less random manner with other groups on the polypeptide chain, and that on the average the different tryptophan residues in denatured proteins have a similar type of environment.     

Measurements of fluorescence lifetimes by use of a hybrid time-correlated and multifrequency phase fluorometer

Measurements of homogeneous and heterogeneous fluorescence intensity decays using a hybrid time-correlated single photon counting/multifrequency phase fluorometer are reported. A trio of fluorophores exhibiting a range of decay profiles was selected. p-Terphenyl, 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene [(Me)2POPOP], and p-bis[2-(5-phenyloxazolyl)]benzene (POPOP), commonly used reference fluorophores, were analyzed initially; their emissions were characterized by monoexponential decay functions. Additionally, emissions from two single tryptophan proteins with different decay profiles were measured. Scorpion neurotoxin variant 3 required three exponentials to fit the emission decay properly (average lifetime approximately 500 ps). At pH 5.5, the fluorescence emission of ribonuclease T1 showed a monoexponential decay with a measured lifetime of approximately 4.0 ns. Thus, in each case, the results from both measurements were consistent between the two detection systems, confirming the view that the two approaches for measuring fluorescence lifetimes are equivalent.     

Influence of solvents and leucine configuration at position 5 on tryptophan fluorescence in cyclic enkephalin analogues

The fluorescence decay of tryptophan is a sensitive indicator of its local environment within a peptide or protein. In this study we carried out fluorescence measurements of the tryptophan residue of cyclic enkephalin analogues of a general formula X-c[D-Dab(2)-Gly(3)-Trp(4)-Y(5)] where X = Cbz or H and Y = D- or L-Leu, in four solvents [water, methanol, acetonitrile, and dimethyl sulfoxide (DMSO)]. An analysis of the tryptophan fluorescence decays using a discrete-exponential model indicates that tryptophan fluorescence decay can be described by a double exponential function in all solvents studied. Lifetime distribution analysis yields a bimodal distribution in protic solvents (water and methanol), whereas an asymmetric, unimodal distribution in an aprotic solvent (DMSO) and uni- or bimodal distributions in acetonitrile solution, depending on leucine configuration. The data are interpreted in terms of the rotamer model, in which the modality and the relative proportions of the lifetime components are related to the population distribution of tryptophan chi(1) rotamers about the C(alpha)--C(beta) bond. The chirality of the Leu(5) residue and solvent properties affect the local environment of the tryptophan residue and therefore influence the distribution of side-chain rotamers. These results are consistent with the results of theoretical conformational calculations.     

A model for multiexponential tryptophan fluorescence intensity decay in proteins.

Tryptophan fluorescence intensity decay in proteins is modeled by multiexponential functions characterized by lifetimes and preexponential factors. Commonly, multiple conformations of the protein are invoked to explain the recovery of two or more lifetimes from the experimental data. However, in many proteins the structure seems to preclude the possibility of multiple conformers sufficiently different from one another to justify such an inference. We present here another plausible multiexponential model based on the assumption that an energetically excited donor surrounded by N acceptor molecules decays by specific radiative and radiationless relaxation processes, and by transferring its energy to acceptors present in or close to the protein matrix. If interactions between the acceptors themselves and back energy transfer are neglected, we show that the intensity decay function contain 2N exponential components characterized by the unperturbed donor lifetime, by energy transfer rates and a probability of occurrence for the corresponding process. We applied this model to the fluorescence decay of holo- and apoazurin, ribonuclease T1, and the reduced single tryptophan mutant (W28F) of thioredoxin. Use of a multiexponential model for the analysis of the fluorescence intensity decay can therefore be justified, without invoking multiple protein conformations.     

Temperature dependence of tryptophan fluorescence lifetime in aqueous glycerol and trehalose solutions

The temperature dependences of tryptophan fluorescence decay kinetics in aqueous glycerol and 1 M trehalose solutions were examined. The fluorescence decay kinetics were recorded in the spectral region of 292.5–417.5 nm with nanosecond time resolution. The kinetics curves were approximated by the sum of three exponential terms, and the spectral distribution (DAS) of these components was determined. An antisymbatic course of fluorescence decay times of two (fast and medium) components in the temperature range from –60 to +10°C was observed. The third (slow) component showed only slight temperature dependence. The antisymbatic behavior of fluorescence lifetimes of the fast and medium components was explained on the assumption that some of the excited tryptophan molecules are transferred from a short-wave-length B-form with short fluorescence lifetime to a long-wavelength R-form with an intermediate fluorescence lifetime. This transfer occurred in the indicated temperature range.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Pulse fluorometry of cyclo-(glycyl-L-tryptophyl).

The fluorescence of cyclo-(glycyl-L-tryptophyl) in trimethyl phosphate has been studied in a temperature range varying from room temperature to -85 degrees C. At room temperature, the fluorescence decay is the sum of two exponentials, the relative amplitude of which depends on the emission wavelength. This can be explained by the presence of the two following emitting molecular states: on one hand the unfolded state, the fluorescence decay time and the emission spectrum of which are close to these of skatole; on the other hand the folded state which has a shorter decay time and a blue-shifted spectrum. By lowering the temperature, the fluorescence spectrum shifts to the blue, while the skatole spectrum shifts to the red. This behavior corresponds to an increase of the folded conformation concentration in agreement with the NMR results. Furthermore the rate of exchange between the folded and the unfolded conformations decreases. Accordingly the wavelength dependence of the fluorescence decay lessens. There are two possible values of the conformational angle x2 differing by 180 degrees, which correspond to the folded state; due to the indole asymmetry, the interactions between the indole and diketopiperazine rings differ in these conformers. Consequently the fluorescence decay remains biexponential even at -85 degrees C.     

Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence.

Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.     

Effect of unfolding on the tryptophanyl fluorescence lifetime distribution in apomyoglobin

Proteins exhibit, even in their native state, a large number of conformations differing in small details (substates). The fluorescence lifetime of tryptophanyl residues can reflect the microenvironmental characteristics of these subconformations. We have analyzed the lifetime distribution of the unique indole residue of tuna apomyoglobin (Trp A-12) during the unfolding induced by temperature or guanidine hydrochloride. The results show that the increase of the temperature from 10 to 30 degrees C causes a sharpening of the lifetime distribution. This is mainly due to the higher rate of interconversion among the conformational substates in the native state. A further temperature increase produces partially or fully unfolded states, resulting in a broadening of the tryptophanyl lifetime distribution. The data relative to the guanidine-induced unfolding show a sigmoidal increase of the distribution width, which is due to the transition of the protein structure from the native to the random-coiled state. The broadening of the lifetime distribution indicates that, even in the fully unfolded protein, the lifetime of the tryptophanyl residues is influenced by the protein matrix, which generates very heterogeneous microenvironments.     

Monitoring gramicidin conformations in membranes: a fluorescence approach

We have monitored the membrane-bound channel and nonchannel conformations of gramicidin utilizing red-edge excitation shift (REES), and related fluorescence parameters. In particular, we have used fluorescence lifetime, polarization, quenching, chemical modification, and membrane penetration depth analysis in addition to REES measurements to distinguish these two conformations. Our results show that REES of gramicidin tryptophans can be effectively used to distinguish conformations of membrane-bound gramicidin. The interfacially localized tryptophans in the channel conformation display REES of 7 nm whereas the tryptophans in the nonchannel conformation exhibit REES of 2 nm which highlights the difference in their average environments in terms of localization in the membrane. This is supported by tryptophan penetration depth measurements using the parallax method and fluorescence lifetime and polarization measurements. Further differences in the average tryptophan microenvironments in the two conformations are brought out by fluorescence quenching experiments using acrylamide and chemical modification of the tryptophans by N-bromosuccinimide. In summary, we report novel fluorescence-based approaches to monitor conformations of this important ion channel peptide. Our results offer vital information on the organization and dynamics of the functionally important tryptophan residues in gramicidin.