Protein fluorescence quenching by small molecules: Protein penetration versus solvent exposure

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Protein fluorescence quenching by small molecules: Protein penetration versus solvent exposure
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  PROTEINS: Structure, Function, and Genetics 1: 109-115 (1986) z Protein Fluorescence Quenching by Small Molecules: Protein Penetration Versus Solvent Exposure Dorothy B. Calhoun,' Jane M. Vanderkooi,' Gary zyxwv . Holtorn,2 and S. Walter Englander' zyx Department zyxwvutsrq f Biochemistry and Biophysics and Department of Chemistry, University of Pennysylvania, Ph iladelph ia, Pennsylvania 19104 BSTR CT Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pat- tern of fluorescence quenching exhibited by quench- ing molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves pro- tein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quench- ing, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For sev- eral proteins having tryptophans known to be buried, no quenching occurs. These results are not consis- tent with the view that the kinds of small molecules studied can quite generally penetrate into and dif- fuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quench- ing is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion. Key words: proteins, protein dynamics, tryptophan exposure INTRODUCTION Proteins are by no means rigid, but engage in inter- nal motions of many kinds.14 The amplitude and time scale of internal protein motions may be deci- pherable from the rates at which agents of varying molecular sue can reach tryptophan side chains and quench their fluorescence or phosphorescence. Such encounters may reflect a deep penetration of the quencher into the protein matrix, a protein unfolding reaction that transiently exposes the tryptophan, or more simply some degree of tryptophan exposure to solvent in the native protein. The latter possibility is especially suggested by the fact that very few protein tryptophans are fully inaccessible to solvent5Y6 The surprising possibility that zyxwvut 2 might penetrate easily into proteins was first suggested by the studies of Lakowicz and Weber, which showed that O2 can zyxwv   986 ALAN R. LISS, INC. quench the fluorescence of many protein tryptophans at nearly diffusion-limited rates. The Lakowicz-We- ber thesis was challenged by othersg but was con- firmed by Calhoun et a1.:who showed that O2 can indeed penetrate rapidly to the positions of some truly buried tryptophans. The possibility that more sizable agents like acryl- amide may similarly penetrate into the protein ma- trix was raised by Eftink and Ghiron (reviewed in reference 11 . In pursuing these issues we have adopted the hypothesis that the rate of true penetra- tion reactions should be sensitive to molecular char- acteristics of the quencher such as sue, polarity, and charge. This hypothesis seems self-evident in light of present knowledge concerning protein structure and is supported by a molecular dynamics simulation12 which displayed an extreme dependence of protein penetration rate on molecular size. Accordingly this paper describes studies of the fluorescence quenching efficiency of a number of dissimilar molecular agents when directed against a series of protein tryptophans having a wide range of accessibility. MATERIALS ND METHODS Proteins obtained from Sigma Chemical Co. in the highest available grade and used as provided in- cluded honey bee mellitin, LADH, rabbit muscle al- dolase, HSA, and RT1 from Aspergillus oryzue. Parvalbumin was prepared from frozen cod fillets, locally obtained, by a modification of methods previ- ously de~cribed.'~.'~ rozen cod fillets (125 g) were homogenized with Tris buffer, pH 8.7, in a blender, stirred for 1 zyx r nd centrifuged (10,000 rpm for 30 min). A 45-80% acetone cut (OOC) was centrifuged; the pellet was resuspended in Tris buffer at pH 7.6 in 1 mM CaC12, heated rapidly (-5 min) to 60 C, and then immediately cooled and centrifuged. Subsequent steps were at 4°C. The supernate was dialyzed against Materials Received June 11, 1986; accepted July 7, 1986. Address reprint requests to S. Walter Englander, Depart- ment of Biochemistry and Biophysics, University of Pennsyl- vania, Philadelphia, PA 19104. Abbreviations used: AP, alkaline phosphatase; DEAE, dieth- yaminoethyl; EDTA, ethylene diamine tetraacetate; HSA, hu- man serum albumin; LADH, horse liver alcohol dehydrogenase; MEK, methyl ethyl ketone; MVK, methyl vinyl ketone; NATA, N-acetyl tryptophan amide; PV, and PV,,, parvalbumin in absence and presence of calcium; RT1, ribonuclease T1.  110 D.B. CALHOUN ET AL. zyxwv 1.6 zyxwvutsrq M piperazine at pH 5.7 and then chromato- graphed (0.5 mumin) on a DEAE cellulose column (25x43 cm) equilibrated with the same buffer. The column was washed until absorbance at 280 nm fell to 0.03 (ca. 24 zyxwvut r and then eluted with a NaCl gra- dient (800 ml, 0-0.1 M NaC1, 0.5 mumin), with pro- tein detected by absorbance at 280 nm. Peaks of the two parvalbumin species were separated, and the tryptophan-containing species was dialyzed against water and lyophilized. Fluorescence quenching exper- iments were done with parvalbumin either contain- ing its normal bound calcium (PV,) or in the presence of 2 mM EDTA to remove thte calcium (PV,,). Among the quenchers used, acrylamide and MVK were from Sigma and bisacrylamide from Eastman Kodak. HPLC grade acetone, acrylic acid, and other reagent grade chemicals were from Fisher Scientific. The solvent used was 0.01 M sodium phosphate at pH 7.0 in 0.1 M NaCl except for studies with mellitin where a low salt condition was used (0.01 M Tris at pH 8.0) to maintain the unstructured state. Quenching Measurements Steady-state fluorescence spectra and intensities in most of the quenching experiments were measured with a SLM-8000 luorimeter interfaced to a Hewlett- Packard 9825 computer. Oxygen quenching experi- ments were done in a Perkin-Elmer 650 fluorimeter adapted to accept a high-pressure stainless-steel cell chamber previously described? In the quenching ex- periments an excitation wavelength of 300 nm was used to avoid contributions from tyrosine fluores- cence. Emission was measured at either 325 or 350 nm depending on the protein in order to avoid the Raman peak (at 334 nm). Protein concentration was usually adjusted to provide optical density -0.1 at 300 nm. Since some of the qenching agents, used at high concentration, have significant absorbance at the ex- citation and emission wavelengths, inner filter cor- rections to the fluorescence intensity data were necessary, as previously described.15 Minor fluores- cence background due to some of the quenchers was measured in parallel experiments set up to replicate the protein experimental conditions, but without pro- tein present. These values were subtracted from the protein data before applying inner filter corrections. Data Analysis Values for quenching rate constant , listed in Table I were obtained from steady-state fluorescence intensity measurements, as is true of almost all the work leading to the penetration hypothesis tested here. Quenching rate constants were computed from the Stern-Volmer equation (Eq. 1 . Here Fo represents steady-state fluorescence inten- sity in the absence of quencher and F in the presence of quencher at concentration [ I. The parameter Fa 1 often increases linearly with [Q], in which case the rate constant kq can be obtained from the (initial) slope of a linear Stern-Volmer plot (Figs. 1- 3). It can be noted that measurements like these do not distinguish dynamic collisional quenching from quenching due to ground state complexation. This does not reflect on the thesis of this paper. If the results found here are to be explained in terms of ground state complexation, this by definition agrees with the conclusion reached, that the quenching ob- served does not represent rapid dynamic penetration. Experiments dealing directly with this issue will be presented elsewhere. RESULTS Studies were done to measure the ability of various small molecule agents to quench the singlet excited state of a number of protein tryptophans. Typical quenching data are exhibited in Figures 1-3, and results are summarized in Table I. TABLE I. Fluorescence Quenching Constants (M-'ns-')* Quencher NATA Mellitin PV,, LADH PV, RT1 Aldolase z 2 zyxwvutsrqpon 5 5 3 2 2 3 Nitrite 13 40 8 2 4 4 2 MVK 13 4 4 3 2 1.5 -0 Nitrate 10 23 0.5 zyxwvu  4 0 2 -0.01 -0.01 Acrylate 3 1 0.7 0.3 0.06 -0 Acrylamide 9 4 2 0.8 0.1 0.25 0.06 Acetone 4 2 0.7 0.6 0 1 -0.02 0.04 MEK 5 3 0 9 0.6 0 2 0.07 -0.04 *Quenching rate constants, listed in units of M-lns- , were measured by decrease in fluorescence intensity as a function of quencher concentration. Fluorescence lifetimes used to calculate the quenching constants were NATA-2.8 ns; mellitin-3.1 nsZ4; V,,-4.5 ns; LADH-6.9 ns3*; PV,4 ns; RT1-3.4 ns; aldolase-2.3 ns? The proteins generally display a multicomponent decay; the lifetime used in each case represents a qualitative average value that reasonably represents, for present purposes, the major fraction of the emitted intensity. For LADH, which has two tryptophans per subunit, Stern-Volmer plots plateaued at high quenching and k, was computed for the quenchable fraction. In experiments with aldolase, protein Concentration was - 1 mg/ml.  PROTEIN FLUORESCENCE QUENCHING 111 z 2 zyxwvutsrqp 0 0 zyxwvutsrqp 0 zyxwvutsrqp 1 02 0 03 4 [Methyl vinyl ketone] zyxwvutsr ig. 1. Stern-Volmer plot of fluorescence quenching by MVK, measured by decrease in steady-state emission intensity. The fluors studied are represented by the following symbofs: 0 ATA; zyxwvut   ellitin; 0 PV  , PV,; 0 T1; 0 ldolase. 40/ / z   30 O F 01 02 zyxwvutsr 3 04 [Acetone] Fig. 2. Stern-Volrner plot of fluorescence quenching by ace- tone, measured by decrease in steady-state emission intensity. Symbols are as in Figure 1. 16 Fo F 13 10 zyxwvutsr OJ 02 03 04 [Methyl ethyl ketone] Fig. 3. Stern-Volmer plot of fluorescence quenching by MEK, measured by decrease in steady-state emission intensity. Sym- bols are as in Figure 1. Fluors and Quenchers Studied Among the fluors studied, we used the fully exposed indole of N-acetyl tryptophan amide (NATA) to iden- tify a number of highefficiency quenchers, with rate constants at or near the diffusional limit. Proteins covering the whole range of tryptophan accessibility were studied, with emphasis on those that appear to have the most protected indoles in order to maximize the chance of observing true protein penetration re- actions. These include the proteins considered by Ef- tink Ghiron to display penetration-dependent fluorescence quenching, except for human serum al- bumin, which is well known to bind agents like the quenchers studied here. Several classes of quenching agents were studied. The small apolar molecule, zyx 2, quenches the fluores- cence of fully exposed tryptophan at a diffusion - limited rate and has been found 7*9, to quench a vari- ety of protein tryptophans at nearly the same rate, indicating an ability to penetrate rapidly into pro- teins. This holds also for the proteins studied here (Table I). The results with 02 can be taken as an indication of the quenching behavior to be expected for agents that can penetrate easily into proteins. That is, incoming 02 s insensitive to molecular bar- riers that effectively block the larger quenchers (Ta- ble I). The resonance energy transfer agents, MVK and NO2- anion, have significant optical absorbance overlapping the indole fluorescence emission band, leading to a small Forster transfer parameter, Rot of about 10 A.15 MVK and NOz can quench trypto- phan fluorescence from a distance of several angs- troms, and this contributes to their quenching capability. These agents are chemical analogs of the contact quenchers used (see below), but they maintain much higher kq values against the less accessible tryptophans (Table I . This is zyx o in spite of their small Forster parameters (10 A), providing evidence that all the tryptophans studied extend quite close to the protein surface. Given the results with 0 nd the transfer agents as background, we are here mainly interested in the possible ability of molecules like acrylamide to pene- trate into and diffuse rapidly within proteins. To study this we used a series of small molecule contact quenchers with varying size, polarity, and charge, which can be expected to affect their penetrational capability. These include acrylamide itself; the ke- tones methyl methyl ketone (acetone) and MEK; ac- rylate, which is a charged analog of acrylamide; and the smaller nitrate anion. These quenching agents have negligible spectral overlap with tryptophan emission and require essentially a van der Waals contact with the target tryptophan in order to quench the tryptophan excited state. On contact, quenching processes in addition to dipolar Forster transfer may act.16J7 Exposed Tryptophan: NATA and Mellitin Against NATA, a freely exposed indole model, the rapidly diffusing 02 molecule displays a diffusion- limited quenching rate constant of 15 M-lns-' (Table I). A number of larger agents show similar k, values (Table I), close to the diffusional limit.  112 D.B. zyxwvuts ALHOUN ET AL. Mellitin (Table I), a 26-residue polypeptide with one tryptophan, was studied at low concentration zyxwv - 1 mg/ml) in low salt conditions to ensure the mono- meric, random coil state18 and major exposure of the tryptophan residue. The nonionic quenchers show similar quenching rate constants against mellitin, about one-half that found for the freely exposed NATA fluor (Table n. The anionic quenchers, nitrite and nitrate (and iodide, not shown), display exaggerated quenching ability, exceeding by severalfold the diffu- sion-limited rate seen for NATA, presumably due to charge attraction since the single tryptophan in mel- litin is adjacent to a sequence of positively charged residues (see also reference 31). Moderately Accessible Tryptophans-PV and LADH The proteins studied are listed in Table I from left to right in order of decreasing sensitivity to quench- ing. Mellitin and PV have a single tryptophan resi- due. Parvalbumin was studied with bound calcium ion (PV,) and in the presence of EDTA (PV,) which chelates the structural calcium of PV, making its tryptophan residue more accessible. LADH has one relatively exposed tryptophan residue per subunit?,19 which is responsible for the quenching observed, and one well-buried tryptophan not accessible to these quenchers (see below). This produces concave down- ward Stern-Volmer plots for LADH quenching. In this case k, values for the more accessible tryptophan can be computed from Lehrer inverse plot^.^^,^ The contact quenchers-NOS-, acrylate, acrylam- ide, acetone, and MEK-all display very similar abil- ity to reach any given tryptophan (Table I) in spite of the fact that the quenchers differ considerably in size and charge. A narrow range is seen for PV,, which is a step down in accessibility from mellitin. Results for the more accessible of the two LADH tryptophans are tightly grouped with k, for the different agents all between 0.4 and 0.8 M-lns-'. Published results show that bisacrylamide, effectively a double-sized acrylamide molecule, has a nearly identical k, of 1 In the presence of calcium (PV,), the sin- gle tryptophan in PV becomes distinctly more pro- tected, but again, all the contact quenchers reach this tryptophan with k, in a tight range, about 0.2 M-lns-', approaching the measurable limit. In summary, taking NATA as a fully exposed stan- dard, the apparent protection factor against the con- tact quenchers for the proteins so far discussed is roughly 2 for mellitin, zyxwvut   or PV,, 10 for LADH, and 40 for PV,. In each protein, the kq values found for the very different contact quenchers are impressively similar in spite of their different sizes and even though some (nitrate, acrylate) are ionic. This is not the pattern expected for a penetrational pathway. The fact that a given tryptophan exhibits similar accessibility to quite different quenchers suggests that ,-lns-l 15 . the tryptophan is still partially exposed at the protein surface. To search for significant protein penetration, it therefore appears necessary to study tryptophans with still lower accessibility, i.e., with k, less than 2 x lo8 M-ls-'. Only a few known proteins produce k, val- ues that low. Nonexposed Tryptophans The single tryptophan in azurin is known from x- ray diffraction studiesz1 o be fully buried. Eftink and Ghiron have shown that fluorescence of the azurin tryptophan is not measureably quenched by acrylam- ide. We confirm this result. Other proteins known to have a well-buried rypto- phan have also some more accessible tryptophans, which renders sensitive fluorescence quenching mea- surements of the buried tryptophan difficult. How- ever, a related approach, the time-resolved measurement of room temperature zyx hosphorescence quenching? which focuses specifically on well-pro- tected tryptophans, then becomes most useful since only the buried tryptophan contributes to the mea- sured phosphorescence. We have LADH and zyxw P which show remarkably long lifetimes for room temperature phosphorescence (0.35 s and 1.5 s respectively). A variety of contact quenchers includ- ing some used here can quench the phosphorescence of the buried tryptophan in LADH and in AP ut this occurs with rate constants that are many orders of magnitude below those measured here (ca. and lo- M-lns-', respectively; measurable15 on the much slower phosphorescence ime scale). In short, studies of the truly buried tryptophans of azurin, LADH, and AP unambiguously rule out fast protein penetration by the kinds of small molecules studied here. It can be noted that 02 reaches these tryptophans with a normal rate constant, - o9 M-ls-'.' Thu s the blockage of the other contact quenchers does not appear to reflect some atypical structural barrier, but merely the fact of full trypto- phan burial within the protein. Tryptophans With Questionable Exposure: RT1 and Aldolase The known repertoire of proteins with quenching rate constants smaller than 0.2 M-lns-' but still measurably greater than zero include only RT1 and aldolase. Data on the quenching of these two proteins by acrylamide supply a major part of the evidence previously interpreted as demonstrating the penetra- bility of acrylamide into proteins. Results for the quenchers we tested are in Table I. 02 quenches RT1 and aldolase fluorescence about as well as other proteins. Quenching by the resonance energy transfer agent, NOz-, suggests that the RT1 and aldolase tryptophans are still within a few angs- troms of the surface, though the k, for MVK suggests  PROTEIN FLUORESCENCE QUENCHING zyxwv 13 some decreased accessibility, especially in aldolase. Acrylamide quenches RT1 with an apparent k, value of 0.25 M-'nspl and aldolase with a kq of 0.1 M-lns-' (measured from the initial part of an up- ward curving Stern-Volmer plot, Fig. zyxwvu ). These results duplicate the earlier findings of Eftink and Ghir~n.~~ The other contact qenchers studied have kqs for these proteins differing only marginally if at all from zero. Clearly the contact quenchers do not penetrate into these proteins at a rate near the free-solvent diffu- sional limit. Various mechanisms for the enhanced activity of acrylamide against RT1 and aldolase (in comparison with the other contact quenchers) can be considered. If we view the immediate tryptophan environment in the sense of solvent, the significant effect of the local surround on quenching efficiency, described by Eftink and Ghir01-1:~ may conceivably exercise a selective effect on the varying quenchers. Another interesting possibility is suggested by the ideas of Be1-8~ nd Berg and von Hippe126 on protein surface diffusion. Perhaps acrylamide can bind nonspecifically to the protein surface and diffuse over it to produce a more efficient encounter with the occluded tryptophan side chain. In the case of aldolase, a possible contributing factor rests on its tendency to dissociate into mono- mers. Rabbit muscle aldolase is a tetramer of nearly identical subunits, each having three tryptophan res- zyxwv 2 .c zyxwvut FO zyxwvutsrqponmlkji   1.6 0.5 I .o I .5 [Acrylamide] Fig. 4. Stern-Volmer plot showing fluorescence quenching of aldolase by acrylamide, measured by decrease in steady-state emission intensity. Concentration of aldolase was zyxwvut .08 mglml zyxwvutsr 0), .3 mglml O), nd 1.4 mglml 0). id~es.~~ tellwagen and Schachman2' have shown that aldolase dissociates at slightly acid pH, being half-dissociated at pH 5.5. Figure indicates that aldolase becomes more susceptible to quenching at lower protein concentration, consistent with a contri- bution from protein dissociation. A structural effect due to weak acrylamide binding is suggested by the remarkably sharp upsweep in quenching at high ac- rylamide concentration seen in Figure 4. Perhaps acrylamide promotes aldolase dissociation and a con- sequent exposure of its tryptophans. Eftink and Ghiron22 noted the ability of acrylamide to bind to aldolase at the high concentrations used in these ex- periments and found some denaturation-like changes above 0.5 M acrylamide. The aberrant interaction of acrylamide with RT1 and aldolase is interesting and deserves further investigation. For the questions addressed in the present study, the near-zero quenching ability found for the other contact quenchers studied seems most indicative. Whatever other undescribed events may be occur- ring, this result is clearly inconsistent with the thesis that such molecules can penetrate rapidly into proteins. DISCUSSION A great deal of discussion has now been devoted to the apparent plasticity of proteins. One possible way to study these issues is by observation of the move- ment of small molecules into and through proteins. This approach is in some ways analogous to the obser- vation of molecular diffusion in lipid bilayers that earlier helped to define the liquid-like character of biomembranes. As a first step one wants to discover the kinds of molecules that can move into proteins and to measure their rates of intrusion and internal migration. It is now clear that molecular oxygen can penetrate, or at least insert, into proteins and quench truly buried tryptophans at a rate only a little lower than the free solvent diffusional limit.7*9s10 an other molecules do the same? Prior Results The possibility that larger molecules like acrylam- ide might move rapidly into and through proteins has been reviewed by Eftink and Ghiron. The major evidence adduced in favor of this conclusion involves the temperature and viscosity dependence of the quenching of a number of proteins by acrylamide. Measured temperature dependences indicate activa- tion energy for the quenching process ranging from 2 to 11 kcal M-l for various proteins, compared to about 4 kcal for the fully exposed NATA model. Inter- pretation of this small range of values in terms of molecular mechanism is problematic and is compli- cated by the possible intervention of various phenom- ena such as protein-quencher binding, especially at the high quencher concentrations necessary for these experiments ( .1-1 M . The observation of a corre- Iation between decreasing k, values among various
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