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Experimental Section

Determination of Distances between Chromophores in Proteins. A Resonance-Energy Transfer Experiment

G U S T A V O G O N Z A L E Z

Secci6n Bioquimica, Instituto de Quimica Universidad Catdlica de Valparaiso Casella 4059, Valparaiso Chile

Introduction When a molecule absorbs electromagnetic radiation it is raised to excited electronic states. In many instances, the excitation energy is lost as heat to the surroundings as the molecule returns to the ground state. However, in many cases this process occurs with the emission of radiation. This is called fluorescence. Substances which display significant fluorescence generally possess chemical groups with delocalized electrons formally present in conjugated double bonds. Nearly all proteins display fluorescence in the ultraviolet region due to the aromatic amino acid residues tryptophan, tyrosine and phenylalanine. In pro- teins containing the three amino acids the fluorescence spectrum is dominated by tryptophan.

Background The lifetime of a molecule in the excited state is about 10 9 s and depends on competition between the radiative emission and any radiationless process, such as the transfer of the excitation energy to the surrounding medium. These nonradiative pro- cesses provide an alternative mechanism for the excited mol- ecules to relax back to the ground state, and their presence will result in a diminution or quenching of the fluorescence intensity.

Fluorescence quenching can also occur by the transfer of the energy of the excited state of a molecule or chemical group (donor) to another molecule or chemical group (acceptor). This transfer occurs without the appearance of a photon, and is primarily a result of interactions between the donor and acceptor. The energy donor returns from the excited state to the ground state and the energy acceptor is simultaneously excited from its ground to its excited state. If the acceptor fluoresces, the original excitation energy reappears as acceptor emission. When the acceptor is nonfluorescent, the transferred energy is disi- pated by non-radiative processes.

The efficiency for depopulation of the excited state by resonance energy transfer depends upon the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor and is given by the expression

E = 1 - (Qt/Qd) (1)

where Qt and Qo represent the quantum yields (fraction of excited molecules that becomes deexcited by fluorescence) of the donor in the presence and absence of resonance energy transfer respectively.

F6rster ] has explained energy transfer in terms of the relative orientation of the donor and acceptor transition dipoles, and the distance between these molecules or groups. It is this latter, dependence upon distance which has resulted in the widespread use of energy transfer to measure distances between donors and acceptors. The energy of this interaction depends on 1/R 3, where R is the distance between donor and acceptor. The rate of energy transfer is proportional to the square of this interaction and hence to 1/R 6 and is given by the expression

E = Roo/(R 6 + R06) (2)

where R0 is a constant for each donor-acceptor and is defined as the distance at which the energy transfer is 50% efficient. From a measurement of the donor quantum yields in the presence and absence of the acceptor (Qt/Qd), R may be calculated if R0 is known.

The prospective user of the energy transfer technique must find a suitable donor-acceptor pair. The first step is to ascertain whether the macromolecule has an intrinsic chromophore that can serve as a donor or an acceptor. A single tryptophan residue in a protein is an excellent donor because its emission, centered at around 330 nm, overlaps the absorption of many potential energy acceptors. Fluorescent coenzymes and substrates such as NADH, FMN and pyridoxal phosphate are also attractive potential donors and acceptors. The heme group in heme proteins is invariably a good acceptor because it has an absorption band extending over the entire visible region, but it is not fluorescent and hence cannot serve as a donor.

Other approaches to obtain donor-acceptor pairs are to use fluorescent analogs (Table 1) or to covalently insert suitable fluorescent groups to the protein. 2 The spectral characteristics of protein-conjugated dyes often permit quite efficient long-range energy transfer from the aromatic amino acids of the protein to the ligands, and also among the ligands themselves.

Resonance energy transfer is of considerable interest as a tool for the study of conformational changes in proteins. Since the efficiency of transfer depends on the inverse of the sixth power of the distance between donor and acceptor chromophores, a conformational change in protein may be measured even if it is caused by only a small change in the distance between two protein-bound chromophores.

Experimental The experiments described here give the procedure to measure the distance between the single tryptophan (donor) of horse- radish peroxidase (HRP) and the heme group (acceptor) located at its active site, based on the observation that the tryptophan fluorescence spectrum overlaps with the heme absorption spectrum (Fig 1).

Absorption spectra were measured with a Varian Cary 219 spectrophotometer. Fluorescence spectra were obtained in a Spex Fluorolog spectrofluorometer. The excitation wavelength was 295 nm to selectively excite the tryptophan residue in HRP.

7

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R

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300 350 400 450

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Figure 1 Overlap of spectra

BIOCHEMICAL EDUCATION 22(3) 1994

Table 1 Fluorescent analogs of biomolecules

Biomolecule Fluorescent analog

ATP, NAD, FAD, CoA and other adenine nucleotides

Heme

Thiamine diphosphate Phospholipids

Ca ~+ Zn 2+

ethenoadenosine derivatives benzoadenosine derivatives formycin derivatives protoporphyrin zinc and tin protoporphyrin thiochrome diphosphate fluorescent derivatives of phosphatidyl ethanolamine Tb 3+ and Eu 3+ Co 2

The absorbance of the solutions at this wavelength was 0.05 or less to minimize the ' inner filter' effect. 3 HRP (type VI) was purchased from Sigma and L-tryptophan from Merck. Apo-HRP was prepared according to the method of Teale. 4 To an ice-cold salt-free HRP solution containing sufficient 0.1 M HCI to give pH 2, is added an equal volume of ice-cold methylethylketone and the mixture is shaken for a short time. On standing separation takes place into a organic supernatant containing all the heme, and a lower aqueous layer containing all the protein, which is freed from dissolved ketone by dialysis against water or by G-25 gel filtration.

Measurement of transfer efficiency, E Calculation of the quantum yield of donor (tryptophan) in the presence of acceptor (heme group) or Qt. This is obtained from the area under the fluorescence spectrum curve of HRP which is equivalent to the sum of the fluorescence intensity at each wavelength in the corresponding wavelength range (Table 2a) and is equal to 632.2.

Calculation of the quantum yield of donor (tryptophan) in the absence of acceptor (heme group) or Qd. This is obtained from the area under the fluorescence spectrum curve of apo-HRP which is equivalent to the sum of the fluorescence intensity at each wavelength in the corresponding wavelength range (Table 2b) and is equal to 4245.5.

Quantum yield values obtained above are relative, and therefore to perform the calculations correctly HRP and apo- HRP solutions must be of identical concentrations. With the use of eqn 1 a value of E = 0.85 was found for the transfer efficiency between tryptophan and the heme group in HRP.

Calculation of R The procedure involves the use of the energy transfer efficiency value previously found and the value of R0. R0 may be obtained from the expression

Ro = 9.79 × 102 (Jvn-4k2Qd)l/6

where n is the refractive index of the medium, Qd is the quantum yield of donor in the absence of the acceptor, U is the orientation factor between the donor and acceptor electric transition dipole moments, and J is the integral of the spectral overlap of the absorption spectrum of the acceptor and the emission spectrum of the donor. A calculation of R0 is beyond the scope of the practical, however a relatively accessible approach to this can be found in Campbell and Dwek. 5

For some typical donor-acceptor pairs which occur in proteins, R0 can be obtained from the literature. 6 For the tryptophan-heme pair a value of 24 A has been reported. By combining this value with the value of the energy transfer efficiency previously found it can be calculated that R, trypto- phan-heme distance in HRP is 18/~.

Table 2 Fluorescence of donor in the presence and absence of acceptor

151

Wavelength HRP Apo-HR P (nm) Intensity Intensity

310 5.5 27.0 312 6.2 30.0 314 7.6 36.0 316 9.0 46.0 318 10.3 57.0 320 12.4 66.0 322 13.8 78.0 324 15.9 90.0 326 17.9 100.0 328 20.0 115.0 330 22.1 126.0 332 24.1 131.0 334 26.1 140.0 336 28.3 143.0 338 29.7 149.0 34(1 31.0 152.0 342 31.7 153.0 344 31.0 154.0 346 29.7 153.0 348 27.6 152.0 350 26.2 150.0 352 24.1 148.0 354 22.1 146.0 356 20.0 142.0 358 18.6 136.0 360 17.2 131.0 362 15.2 124.0 364 13.8 117.0 366 12.4 110.0 368 11.0 104.0 370 9.7 98.0 372 8.3 94.0 374 6.9 88.0 376 5.5 83.0 378 4.8 78.0 380 4.1 75.0 382 3.9 72.0 384 3.4 68.0 386 3.2 66.0 388 2.9 60.0 39O 2.8 57.5

Sum = 632.2 Sum = 4245.5

In summary the experiments described here offer a simple and inexpensive procedure for the introduction of the resonance- energy transfer concept and the determination of the distance between two chromophores in a protein.

Reference I F6rster, T (1948) Ann Physik 2.55-75

2Stryer, L (1978) Ann Rev Biochem 47, 819-846

3Montero, M T, Hernandez, J and Esterlich, J (1990) Biochem Ed 18, 99-101

4Teale, F W J (1959) Biochim Biophys Acta 35, 543

5Campbell, I D and Dwek, R A (1984) in Biological Spectroscopy, Benjamin/Cummings, Chapter 5

6Steinberg, 1 Z (1971) Ann Rev Biochem 40, 83-114

B I O C H E M I C A L E D U C A T I O N 22(3) 1994