Study of Adrenocorticotropic Hormone Conformation by ... - Europe PMC

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Department of Molecular Biology and Biophysics, Swiss Federal Institute of Technology, 8049 .... Present address: McCollum-Pratt Institute, The Johns Hop-.
Proc. Nat. Acad. Sci. USA Vol. 69, No. 4, pp. 975-979, April 1972

Study of Adrenocorticotropic Hormone Conformation by Evaluation of Intramolecular Resonance Energy Transfer in N-Dansyllysine21ACTH-(1-24)-Tetrakosipeptide (fluorescence spectra/Forster parameters/side-chain flexibility/intramolecular distances)

PETER W. SCHILLER* Department of Molecular Biology and Biophysics, Swiss Federal Institute of Technology, 8049 Zurich, Switzerland

Communicated by Hane Neurath, February 2, 1972 The solution conformation of ACTH was ABSTRACT studied by intramolecular resonance energy transfer and fluorescence depolarization with the synthetic, biologically active, derivative N'-dansyllysine'l-ACTH-(1-24)-tetrakosipeptide. The Forster parameters involved in energy transfer from the donor Trp9 to the dansyl acceptor attached to the side chain of Lys21 were determined from measurements with ACTH fragments containing either the donor or the acceptor alone. From determinations of the fluorescence quantum yields of the donor in the derivative and in the native ACTH-(1-24)-tetrakosipeptide, it was shown that besides energy transfer no additional quenching of the donor fluorescence is introduced by the presence of the acceptor. The short measured rotational relaxation time of the dansyl chromophore reflects the flexibility of the lysine side chain and justifies the use of an average value for the orientation factor in the distance calculations. The calculated intramolecular distance is remarkably independent of solvent, indicating a "random coil" situation with regard to residues 9-21.

Long-range radiationless transfer of singlet excitation energy from an energy donor to an acceptor chromophore can occur over distances of the order of 10-100 X. F6rster's theoretical treatment of the phenomenon (1-3), based on "very weak" dipolar coupling between donor and acceptor, predicts a dependence of the transfer rate on the inverse sixth power of the distance between the two chromophores. This proposition and the early observations of energy transfer between chromophores in proteins by Stryer (4), Weber and Teale (5), and Velick (6) indicated the potential of energy transfer measurement as a means of calculating distances between fluorescent residues in biopolymers in solution. The validity of F6rster's equation has been confirmed py several excellent experiments with donor-acceptor paizs of fixed separation and random orientation (7-9), and he applicability of the energy transfer method to conforma ional studies on macromolecules was further demonstrate by a careful study on a series of flexible model compound /(10). Numerous studies on energy transfer in biopolymers have been reported. In addition to intrinsic fluorescent chromophores, extrinsic fluorophores bound to a specific site in the macromolecule were used in these investigations. However, in almost all the cases, the interpretation in terms of structural

parameters has been complicated by the presence of multiple donors and acceptors and the difficulty of determining all the critical quantities in the Forster equation. This paper describes a study on energy transfer in N'-dansyllysine2l-

ACTH-(1-24)-tetrakosipeptide ([Lys(Dns)21]ACTHlI24): Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-GlyLys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro, 24 I NH

(CH3)2N which has been obtained by synthesis and displays biological activity similar to that observed with the ACTH-(1-24)tetrakosipeptide (ACTH124) (11, 12). This compound offers the possibility of measuring the mean intramolecular distance between one donor (Trp9) and one acceptor Lys(Dns)21 under various conditions. Moreover, the spectral properties of the fluorophores allow for an unambiguous determination of the fluorescence quantum yield of the donor and the orientation factor, the two most critical parameters in the Forster treatment. ,-

MATERIALS AND METHODS

Peptides and Peptide Derivatives. [Lys(Dns)21]ACTHl-24 was prepared (11, 12) by a synthetic route similar to that used for the synthesis of ACTH1l24 (13). The derivative elicited 100% activity with respect to both maximal stimulation and half-effect concentration on membrane adenylate cyclase of adrenal and fat cells (experiments by T. Braun and 0. Hechter) and about 100% maximal stimulation of corticosteroid production and lipolysis at about 10 times the concentration of ACTHI_24 in isolated cells or tissue slices of the adrenal or the epididymal fat pad (experiments by P. Bally, J. Maller, and G. Sayers). The reference compound N-dan-

syllysine2l-ACTH-(11-24)-tetradekapeptide

Abbreviations: ACTH, adrenocorticotropic hormone, Dns (= dansyl), 1-dimethylaminonaphthalene-5-sulfonate. * Present address: McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Md. 21218.

([Lys(Dns)21]-

ACTHn1 24): Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys(Dns)Val-Tyr-Pro, 975

976

Biochemistry: Schiller

Proc. Nat. Acad. Sci. USA 69 (1972)

After introduction of the numerical values for all the physical constants, an expression for the calculation of the interchromophoric distance is either obtained by combination of Eq. [1], [3], and [4]: [8.9= 8[7

X10-28AD..K2. X2 JA n -

4

WT

T

[cm],

[6]

or [1], [3], and [5]:

K2 11/ = [8.79 [cm], [ X 10-2 -JAD-4-D-J AD D T][]

~~~n4

A (m)

FIG. 1. Spectral overlaps in [Lys(Dns)21JACTHi-24. Absorption spectra of Trp (curve 1) and Lys(Dns) (curve 2); fluorescence emission specta of Tyr (curve 3), and Trp (curve 4).

was easily obtained by acidolysis of an intermediate compound used in the synthesis of [Lys(Dns)2"]ACTH1 24. ACTHI424 (Synacthen) was a generous gift of Dr. W. Rittel, Ciba-Geigy AG, Basel, Switzerland. Equations. Following the treatment by Conrad and Brand (10), I distinguish three different processes whereby a fluorophore in its first excited state can loose its energy and return to the ground state: (i) emission of light (fluorescence); (ii) a sum of various other quenching effects (internal conversion, intersystem crossing, etc.); (iii) nonradiative excitation energy transfer to another chromophore. These processes are assumed to be competitive and to occur at rates that are described by the constants kF, kQ, and k7,, respectively. The rate constant, klr, for energy transfer from a donor dipole situated at distance R from an acceptor is given by the F6rster Eq. [1] (1-3): -9 (In 10) K'

l28=6Nn4(RN JAD

[1]

with OD

JAD = f

FD(X)eA(X)X4dX,

[2]

where K = dipole-dipole orientation factor, N = Avogadro's number, n = refractive index of the medium, and T = fluorescence lifetime of the donor (the inverse of kF); JAD = overlap integral between the molar decadic absorption coefficient of the acceptor (XA) and the spectral distribution of the fluorescence of the donor, normalized to unit (FD), modified by the wavelength factor X4. In Eq. [1], kr, and T may be replaced by quantities that can easily be obtained spectroscopically: k '' = kF + kQ + kJr

=

Transfer efficiency

kp. = Fluorescence quantum yield of the donor without energy kp + kQ

[3]

?

17]]

Eq. [7] offers the advantage of taking into account possible additional quenching of the donor fluorescence in the presence of the acceptor (10) (aside from quenching through energy transfer), provided that 'D is experimentally accessible. Such additional quenching effects can be detected by application of Eq. [8]: 'OD = D(1 -'12), [8] which is easily obtained by combination of Eq. [3], [4], and

[5]. Determination of the Forster Parameters. Corrected fluorescence excitation and emission spectra were obtained on an absolute spectrofluorometer (Turner model 210 "Spectro") (14). Absorption spectra were recorded on the same instrument operated as a dual-beam spectrophotometer. Fluorescence polarization spectra were recorded on a recording fluorescence polarization photometer built by Deranleau (15). The fluorescence lifetimes were measured on a pulse fluorimeter constructed by Studer et al. (16). All fluorescence measurements were made at concentrations of the order of 10 ;AM in stoppered 1-cm, square, quartz cuvets; the solutions were equilibrated with nitrogen. Determination of the transfer efficiencies was repeated 2to 4-times in each solvent, with a freshly prepared solution for each measurement. The fluorescence quantum yield of the donor in the absence of energy transfer, 4D, was obtained by comparison of the tryptophan emission of ACTHl-24 in the particular solvent under investigation to that of pure tryptophan, for which an absolute quantum yield in water of 0.14 was assumed.t The same method was applied to determine the quantum yield of the tryptophan fluorescence, OD, in [Lys(Dns)21]ACTH-24, whereby the small contribution of dansyl fluorescence in the region of tryptophan emission was corrected for by comparison with the emission spectrum of [Lys(Dns)21]ACTHII 24. Determination of the spectral overlap integrals was based on the tryptophan emission of ACTH1I-4 and the absorption spectrum of Dns-lysine and was performed by following the usual procedure. Values of the refractive indexes at the mean wavelength of 340 nm were obtained from

the literature (20-22). RESULTS Spectral overlaps and energy transfer in [Lys(Dns)911-

[4]

ACTH,-mI The dansyl group, the tryptophan residue, and the two tyrosine residues are the only chromophores in [Lys(Dns)21]-

= Fluorescence quantum yield [5] OD = kF + kF kQ + k' of the donor with energy

t I use the value of 0.14 for the quantum yield of tryptophan in aqueous solution, based on recent determinations by Chen (17), Borresen (18), and Eisinger (19).