Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

J. Mol. Biol. (2007) 371, 1378–1391

doi:10.1016/j.jmb.2007.06.038

Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel Jianli Lu 1 , William R. Kobertz 2 and Carol Deutsch 1 ⁎ 1

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104-6085, USA 2

Department of Biochemistry and Molecular Pharmacology, UMASS Medical School, Worcester, MA 01605, USA

Electrostatic potentials influence interactions among proteins and nucleic acids, the orientation of dipoles and quadrupoles, and the distribution of mobile charges. Consequently, electrostatic potentials can modulate macromolecular folding and conformational stability, as well as rates of catalysis and substrate binding. The ribosomal exit tunnel, along with its resident nascent peptide, is no less susceptible to these consequences. Yet, the electrostatics inside the tunnel have never been measured. Here we map both the electrostatic potential and accessibilities along the length of the tunnel and determine the electrostatic consequences of introducing a charged amino acid into the nascent peptide. To do this we developed novel probes and strategies. Our findings provide new insights regarding the dielectric of the tunnel and the dynamics of its local electric fields. © 2007 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: MTS reagents; maleimide reagents; cysteine modification; pegylation; translation

Introduction The ribosomal exit tunnel is not simply a passive, innocent conduit. It is a non-homogeneous pathway that dynamically influences protein synthesis.1–6 Although the tunnel is principally lined by rRNA, especially in the region within 15 Å of the peptidyl transferase center (PTC), proteins also protrude into the tunnel7–10 and may modulate peptide synthesis. In addition, hydrophobic interactions may play a role in the dialogue between nascent peptide and the ribosome.6 Water is present in this cavity1,11–13 at the same time that newly synthesized peptide moves through it from the PTC to the exit port, some 100 Å away. The tunnel is 10–20 Å wide,7–10 which has energetic consequences14,15 and dictates a tight squeeze for peptide, water, and dissolved ions. Moreover, narrow clefts and vestibules can focus any electrostatic potentials† within the ribosomal exit tunnel,16–18 and † Electrostatic potential at a specific location within the tunnel can be defined as the work required to move a dimensionless unit charge to this position from infinitely far away. Abbreviations used: PTC, peptidyl transferase center; MAL-ET, maleimide ethyltrimethylammonium; MAL-ES, maleimide ethylsulfonate; MTS, methanethiosulfonate; βME, β-mercaptoethanol. E-mail address of the corresponding author: [email protected]

therefore could affect concentrations of ions such as Mg2+, which is known to interact with RNA and to be a necessary co-factor for ribosome function. Yet, the electrostatic potential inside the tunnel has not been measured. Here, we map experimentally the electrostatic potential along the ribosomal exit tunnel containing a resident nascent peptide. We also assess the electrical consequence of introducing charged amino acids into the nascent peptide inside the tunnel. Is the charge of an amino acid side-chain felt throughout the length of the tunnel? Elucidation of the electrostatic potentials in the tunnel may be critical for understanding folding events, signaling within the tunnel, and arrest mechanisms. For example, arrest signals may involve specific sequence motifs, helix structures, and/or charged side-chains,2,4,5,19–22 all of which could interact with the local electrostatic potential in the tunnel. To map electrostatic potentials inside the ribosomal exit tunnel, we used a previously constructed molecular tape measure,1 cysteine scanned along the tape measure residing in the tunnel, and measured the rate constants for modification of each engineered cysteine by positively and negatively charged cysteine-modifying reagents, namely, small maleimides (MAL). An analogous approach, using methanethiosulfonate (MTS) reagents, has been used to measure electrostatic potentials in ion channel crevices and vestibules.23 However, the underlying assumption that the reactivities of these reagents to cysteine residues, relative to free thiols in

0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

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solution, depend only on electrostatic potential has never been tested directly. Moreover, the MTS reagents, while excellent for electrophysiological studies, are not ideally suited for biochemical probing of the ribosomal tunnel (see below). Novel pairs of probes, expressly synthesized and characterized for this purpose, are necessary. We now introduce and validate the use of oppositely charged maleimides to determine electrostatic potential and report that the potential inside the tunnel is negative, it varies in magnitude along the length of the tunnel, and a charge introduced into the peptide is felt at a specific location only if it is positioned adjacent to the modifiable cysteine. This suggests the tunnel dielectric is relatively high and leads to a new paradigm: during translation, the elongating peptide creates a dynamic wave of electrostatic potentials along the tunnel in accord with the peptide's primary sequence. In addition, we report the accessibility along the entire length of the tunnel, which has implications for the shape of the exit tunnel in the presence of a nascent peptide. Intrinsic electrostatic potentials are critical to conformational folding, kinetics of biochemical reactions, and organizing solvent, ions, and molecules with monopole, dipole, and quadrupole moments.28 Given its central role in biology, the electrostatic potential is likely to be just as important in the ribosome, where it could modulate such fundamental processes as peptide elongation, folding, and signaling events during translation.

Commercially available MTS reagents (Figure 1) have been used for this purpose,30 but have three disadvantages for our experiments. First, MTS reagents hydrolyze quickly, especially methanethiosulfonate ethyltrimethylammonium (MTS-ET, τ½ = 8 min at 0–4 °C; data not shown). Second, MTS adducts contain a disulfide bond and are therefore cleaved by reducing conditions. Third, MTS reagents modify cysteine residues extremely rapidly, severely limiting the accuracy of our kinetic

Results Two strategies were used to measure the electrostatic potential at discrete locations inside the ribosomal tunnel. First, we created nascent peptides that remained attached to tRNA and the ribosome at the PTC. This nascent peptide was engineered to contain a single cysteine residue. Second, we used a quantitative relationship between the rates of modification of an engineered cysteine and the electrostatic potential.29,30 This approach has been used to measure the electrostatic potential inside ion channel pores and crevices.23–27,29 The relative rates of modification of a given cysteine in a protein by positively and negatively charged reagents relative to the ratio of these rates for a free thiol in solution, e.g. β-mercaptoethanol (βME), is an exponential function of the electrostatic potential (Ψ). The equation describing this relationship is: Ucys =UhME ¼ eðzþ z ÞFC=RT

ð1Þ

where ρcys is the ratio of the rate constant for modification of a particular cysteine for a positively charged reagent relative to the modification rate constant of that same cysteine for a negatively charged reagent, z+ and z− are the charges of the two reagents ( ± 1 for the reagents we used), and ρβME is the ratio of rate constants for modification of βME by the same two reagents.

Figure 1. Experimental tools for measuring electrostatic potential in the tunnel. (a) Charged cysteinemodifying reagents. Structures are shown for MTS-ES and MTS-ET (top row) and MAL-ES and MAL-ET (bottom row). The atoms are color-coded as CPK spheres. The structures of the reagents were optimized by ab initio methods (HF/6-31++G*) using Gaussian03 [http://www. gaussian.com] and displayed with DS Viewer Pro [http:// www.accelrys.com]. (b) Cartoon of nascent peptides (not drawn to scale). Each nascent peptide was made from the N terminus of Kv1.3 using either an engineered BstEII site or a natural NcoI restriction site to place cysteine mutations 73C and 72C, respectively, at the indicated positions inside (73C) or outside (72C) the tunnel. The number of residues from the PTC to (and including) the cysteine (ΔPTC) is 27 and 71, respectively. The NcoI-cut peptide is 142 amino acids and the BstEII-cut peptide is 95 amino acids in length.

1380 measurements. To obviate these problems, we synthesized and characterized a new pair of low molecular mass cysteine reagents: maleimide ethyltrimethylammonium (MAL-ET) and maleimide ethylsulfonate (MAL-ES; Figure 1(a)). These reagents differ from MTS-ET and methanethiosulfonate ethylsulfonate (MTS-ES) only with respect to their reactive moiety. MTS reagents undergo a disulfide exchange reaction with cysteine residues to produce a mixed disulfide and a leaving group, methanesulfinate,30 whereas maleimides are Michael acceptors that react with an ionized SH group to produce a stable carbon–sulfur bond. Accordingly, the reaction rates of maleimides with thiol groups are orders of magnitude slower than MTS reagents.30–32 To test these new reagents and to verify that the relative reactivities of oppositely charged reagents are reliable probes of electrostatic potential, rather than say structural differences between the probes, we compared the set of MAL reagents to the set of MTS reagents in the cysteine-modification reaction of a nascent peptide. The pair of MTS reagents serves as a control, since they have been widely used for the purpose of measuring electrostatic potential. The van der Waals volumes of both sets of reagents are similar, e.g. 237 Å3 and 284 Å3 for MAL-ES and MAL-ET, respectively, and 199 Å3 and 286 Å3 for MTS-ES and MTS-ET, respectively. This is especially relevant given the reported range of diameters (10 Å–20 Å) of the ribosomal tunnel. Both classes of reagent will therefore experience similar volume constraints for a given location in the tunnel. Thus, while the reaction mechanisms are quite different for MTS and MAL reagents, their charges and molecular volumes are comparable, rendering these probes ideal for comparison. We made two nascent peptides derived from the N terminus of the T1 domain of Kv1.3, each engineered with a cysteine that is 27 (73CΔ27, BstEII-cut) or 71 (72CΔ71, NcoI-cut) residues from the PTC (Figure 1(b)). Cysteine 73C is located inside the ribosomal tunnel, whereas cysteine 72C is located outside the tunnel.1,33 We treated the nascent peptide first with cysteine-modifying reagent (MTS or MAL) for various times, quenched with reducing agent, and then assessed modification using a pegylation assay of the residual free thiol.31 A cysteine that has not been modified by MTS or MAL reagent during the allotted incubation time will be labeled subsequently with PEG-MAL, a mass-tag used to identify available cysteine sidechains. When PEG-MAL binds covalently to the peptide, it shifts the apparent protein molecular mass by ≥ 10 kDa on a protein gel. As shown in Figure 2 (left panels) for 73CΔ27, the band labeled 0, which represents the nascent peptide that has been previously modified with reagent and is now unavailable for labeling with PEG-MAL, increases with increasing modification time. That is, the availability of the 73C side-chain decreases with time of exposure to MTS reagent. The band labeled 1 represents a cysteine that was not modified with

Ribosomal Tunnel Electrostatics

Figure 2. Time course of cysteine modification using MTS and MAL reagents. After treatment with each of the indicated reagents, 73C, which is located inside the tunnel (Figure 1(b)) in the BstEII-cut nascent peptide (73CΔ27), was pegylated and fractionated on gels as described in Materials and Methods. Numbers to the left of the gels are molecular mass standards (kD); numbers to the right indicate unpegylated (0) and singly pegylated (1) protein. The plot to the right of each gel is the fraction of individual cysteine residues pegylated, which is inversely correlated with the fraction of individual cysteine residues modified by the specified reagent. The fraction pegylated is calculated as radioactivity (c.p.m.) in band 1 divided by the sum of radioactivity in bands 1 and 2. A singleexponential function was fit to the data to give a modification rate constant of 712, 9563, 9.6, and 315 M− 1s− 1 for MTS-ES, MTS-ET, MAL-ES, and MAL-ET, respectively.

MTS reagent and was labeled subsequently with PEG-MAL. The disappearance of band 1 is faster for MTS-ET compared with MTS-ES in spite of the twofold higher concentration of MTS-ES. To get equivalent rates of disappearance of band 1, the corresponding charged MAL reagents must be used at higher concentration due to the marked differences in the intrinsic rates of the two categories of reagents. (NB, in Figure 2, the time scales and concentrations of reagents are different in each depicted modification time course). The disappearance is plotted versus

Ribosomal Tunnel Electrostatics

time of exposure to MTS or MAL reagent and fit with equation (9) (Materials and Methods) to yield a rate constant (Figure 2, right panels). Modification of 72C (outside tunnel) by MTS-ET and MTS-ES yields rate constants of 14.8 × 10 4 M − 1 s − 1 and 1.56 × 10 4 M − 1 s − 1 , respectively. The ratio of these rate constants, ρcys, is 9.5. Modification of 72C by MAL-ET and MAL-ES gives rate constants of 3050 M − 1 s − 1 and 217 M − 1 s − 1 , respectively, yielding a ratio ρcys of 14. The ratio of relative rates of maleimides to MTS reagents is 1.47. Similar measurements and calculations were carried out for 73CΔ27 (inside tunnel) and give ratios of relative

1381 rates of MAL/MTS of 1.52, in spite of the fact that the estimated electrostatic potentials for residues inside and outside the tunnel are quite different (see below). This suggests that the measured ratios of rates are a function of electrostatic potential at the specified cysteine residue, independent of the particular cysteine reagent used. Moreover, the overall rates obtained for each reagent vary linearly with concentration of the cysteine reagent (data not shown), indicating that the modifications are wellbehaved, bimolecular reactions. To calculate the electrostatic potential, we need a reference standard for the reaction of a free thiol in

Figure 3. Cysteine modification in the tunnel. (a) Single-cysteine tape measures. Sequences of all BstEII-cut constructs with either 62C or 73C (red) in different length nascent peptides are shown indicating successive shortening of the chain length, which positions the cysteine closer to the PTC. For each construct, the number of residues from (and including) the cysteine to the PTC is indicated by Δ followed by a number. For example, 62CΔ34, represents a construct containing 62C located 34 residues from the PTC. A bolded Q indicates that a glutamine was substituted for a charged native residue. (b)Time course of modification of 62CΔ20 using MAL reagents. The nascent peptide was treated with either MAL-ET (10 μM) or MAL-ES (100 μM), pegylated, and fractionated on gels as described in the Materials and Methods. The plot to the right of each gel is the fraction of individual cysteine residues pegylated, which is inversely correlated with the fraction of individual cysteine residues modified by the specified reagent. The fraction pegylated is calculated as described in the legend to Figure 2 and a single-exponential function was fit to the data to give a modification rate constant of 262 M− 1s− 1 and 13.7 M− 1s− 1 for MAL-ET and MAL-ES, respectively. A similar set of experiments was carried out for the nascent peptide tape measure containing 73CΔ20. Methods and calculations are as described for 62CΔ20 to give a modification rate constant of 163 M− 1s− 1 and 6.7 M− 1s− 1 for MAL-ET and MAL-ES, respectively.

Ribosomal Tunnel Electrostatics

1382 solution with these reagents. These rates have been determined for MTS reagents reacting with β-mercaptoethanol, and the ratio, ρβME, is 7.00.30 For MAL-ET and MAL-ES, we determined the rates of modification of β-mercaptoethanol using stoppedflow spectroscopic methods (see Materials and Methods). The ratio (MAL-ET/MAL-ES) is 6.99 at ∼ 6 °C, pH 7.3 in the buffer used in all experiments. These values were used in equation (1) to calculate electrostatic potentials. Electrostatic potentials along the ribosomal exit tunnel Having validated the probes and determined the modification rates for β-mercaptoethanol, we next addressed the choice of nascent peptide to use to measure the electrostatic potential at discrete, known locations in the ribosomal exit tunnel. A molecular tape measure that measures the functional length of the ribosomal tunnel accurately was previously characterized in our laboratory1 and was ideal for this purpose. This molecular tape measure is a 95 amino acid nascent chain (attached to the ribosome) composed of the N terminus of Kv1.3. This N terminus includes a portion of the T1 sequence that is known to be all-extended in the mature protein34–36 and has been engineered to reside within the ribosomal exit tunnel.1 A cysteine scan of the tape measure, followed by pegylation, yields a monotonically increasing extent and rate of modification with distance from the PTC.1 We designed a series of constructs containing a single cysteine, 62C, but that differed in the number of residues between the cysteine and the PTC (Figure 3(a)). For each nascent peptide, a deletion was made from the C terminus to shorten the chain by the appropriate number of amino acids to position the cysteine a specified number of residues from the PTC (Δ). For those cysteine residues located near the exit port and/or inside the tunnel, the distance in Å can be estimated because the tape measure alone constitutes the intervening segment between the cysteine and the PTC. For example, in the experiment shown in Figure 3(b), 62C was 20 residues from the PTC, a distance of ∼ 60 Å, and inside the tunnel. We refer to this construct as 62CΔ20. The time course of modification of this cysteine is shown for 10 μM MAL-ET and 100 μM MAL-ES. The corresponding plots of the calculated fraction labeled, along with single-exponential fits to the data, are displayed to the right of each gel. The rate constants for modification of 62CΔ20 are 239( ± 44) and 12.4 ( ± 1.2) M− 1s− 1 (n = 3) for MAL-ET and MAL-ES, respectively. Similar experiments were carried out with other 62C constructs shortened as shown in Figure 3(a) to reside within the tunnel at different locations from the PTC. To ensure that we are measuring the electrostatic potential of the ribosomal tunnel, independently of a particular cysteine and/or its flanking residues, we created another series of constructs using 73C

and strategically shortened the nascent chain so that 73C occupied the same location in the tunnel as 62C in the previous set of constructs (e.g. Δ27, Δ20, and Δ13). Overlap of these constructs permits measurement of three locations using two different cysteine residues with different flanking residues. For example, in construct 73CΔ20, 73C is 20 residues from the PTC, a distance of ∼60 Å, the same location as 62C in 62CΔ20. Modification of 73CΔ20 is shown in Figure 3(b), along with the corresponding single-exponential fits to the data. The rate constants for modification of 73CΔ20 are 181( ± 14) and 7.7( ± 0.8) M− 1s− 1 (n = 3) for MAL-ET and MAL-ES, respectively. As for 62CΔ20, the MAL-ES is slower than MAL-ET. Moreover, for both reagents, a cysteine located within the tunnel is modified about tenfold slower than a cysteine located outside the tunnel. This is consistent with previous results reported for PEG-MAL and indicates a decreased accessibility inside the tunnel.1 The calculated ratio of modification rates, ρcys, can be determined for each cysteine along the tunnel and used to calculate the electrostatic potential, Ψ, at each location (equation (1)). The rate constants for modification of each cysteine residue are given in Table 1, along with calculated ratios and electrostatic potentials. The electrostatic potentials are summarized in Figure 4. Figure 4(a) illustrates the excellent agreement between different cysteine residues at the same location. Figure 4(b) indicates the overall average potential for any given tunnel location. Each overall average potential was obtained by pooling Table 1. Rate constants of modification and electrostatic potentials Rate constant (M− 1s− 1)

Ratio

Electrostatic potential (mV)

k+

k−

ρ

Ψ

62CΔ74 62CΔ34 62CΔ30 62CΔ27 62CΔ20 62CΔ13

2078 ± 64 1781 ± 357 1398 ± 163 455 ± 54 239 ± 44 187 ± 46

199 ± 9 56.3 ± 0.9 N.D. 11.0 ± 0.7 12.4 ± 1.2 6.1 ± 1.5

10.3 ± 0.5 31.7 ± 5.9 N.D. 41.0 ± 2.4 19.1 ± 2.4 31.0 ± 0.8

− 4.9 ± 0.6 − 18.7 ± 2.2 N.D. − 22.1 ± 0.8 − 12.4 ± 1.6 − 18.6 ± 0.3

73CΔ67 73CΔ27 73CΔ20 73CΔ13 73CΔ6

1255 ± 166 390 ± 73 181 ± 14 283 ± 35 84.3 ± 8.2

164 ± 5 10.0 ± 0.8 7.7 ± 0.8 8.8 ± 0.2 6.3 ± 0.8

8.0 ± 0.8 39.3 ± 7.2 23.3 ± 1.7 32.0 ± 2.9 13.7 ± 0.9

− 1.6 ± 1.3 − 21.4 ± 2.2 − 15.0 ± 0.9 − 19.0 ± 1.2 − 8.4 ± 0.8

663 ± 41 602 ± 54 664 ± 131 717 ± 96 301 ± 23 849 ± 66

17.3 ± 0.4 28.8 ± 1.3 9.2 ± 0.5 19.1 ± 1.8 9.2 ± 0.5 21.1 ± 0.4

38.3 ± 3.1 21.0 ± 1.6 72.3 ± 15.1 37.7 ± 4.1 32.7 ± 1.7 40.3 ± 3.1

− 21.2 ± 1.0 − 13.7 ± 1.0 − 28.9 ± 2.9 − 21.0 ± 1.4 − 19.3 ± 0.6 − 21.9 ± 1.0

73CΔ27-Q1 73CΔ27-R1 73CΔ27-D1 73CΔ27-R2 73CΔ27-D2 73CΔ27-R3

Data are mean ± SEM for triplicate samples except for 73CΔ27, n = 4. N.D., not determined. Individual ρ values were calculated for each pair of kinetic experiments. These ρ values were converted to Ψ values according to equation (1) and then used to calculate mean ± SEM for n = 3 or 4 independently determined Ψ values.

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Figure 4. Electrostatic potential map of the ribosomal exit tunnel. (a) Schematic of the tunnel and associated Ψ values in mV. The distances and assignments of Ψ values were made based on the tape measure. The location of each cysteine is indicated by a yellow circle for 62C and a green circle for 73C. The electrostatic potentials measured using each cysteine residue are indicated by a line connecting the number (in mV) to the cysteine used to make that specific measurement. The values are means (n = 3–4) for each cysteine. Means ± SEM are given in Table 1. (b) Electrostatic potential mapped onto the crystal structure of H. marismortui.13 The structure shown in (b) is taken from Voss et al.13 Ribosomal proteins, L4 and L22, are indicated, as are the PTC and the A and P-sites. An additional 20 Å region is added to the structure to account for the longer tunnel reported by others.1,7,8 For illustrative purposes, the Ψ values are shown at a given location as averages (rounded off to whole numbers) of both 62C and 73C values that are listed in Table 1. The electrostatic potentials for adjacent locations, except between 80 Å and 102 Å, are significantly different (p ≤ 0.001, ANOVA, Student-Newman-Keuls method).

the 62C and 73C data (Table 1) obtained at the same location. Three conclusions may be drawn. First, the electrostatic potential inside the tunnel is negative compared with the environment outside the tunnel. The electrostatic potential in the vicinity of cysteine residues that are outside the exit tunnel, e.g. 60–70 residues from the PTC, is only slightly negative and close to zero. Second, the ribosomal exit tunnel is not isopotential, but rather the electrostatic potential varies along the length of tunnel: voltages range from − 8 mV to − 22 mV. This indicates that the dielectric constant in the tunnel is high enough to screen local charges. Third, the ribosomal tunnel is the major determinant of electrostatic potential because both 62C and 73C, each with different flanking sequences, give the same electrostatic potentials for the same location in the tunnel. Change in accessibility along the ribosomal exit tunnel The kinetics of modification can be used additionally to detect changes in accessibility in the

tunnel. According to the formulation by Elinder et al.,27 as described in the Materials and Methods (equation (7)), the ratio of rate constants for a given cysteine by a monovalent cationic reagent (MAL-ET in our study) for two different locations will estimate the n-fold change in accessibility. For this purpose, we analyzed the rate constants for modification of both the 62C series of constructs and the 73C series (Table 1) using equation (7). The ratio for each location was normalized to NcoI-constructs for 62C (Δ74 from the PTC; 62CΔ74) and 73C (Δ67 from the PTC; 73CΔ67), respectively, both of which are well outside the tunnel. Figure 5 shows the n-fold change in accessibility as a function of distance from the PTC. As the chain length was shortened, the accessibility decreased monotonically. For the first ∼ 80 Å from the PTC, the peptide in the tunnel is relatively inaccessible, changing by only ≤ 2-fold. However, over the final 20 Å near the exit port, the accessibility increases and approaches the accessibility of the cysteine in the NcoI-cut construct, which is ≥ 67 residues from the PTC. These last 20 Å correspond to the region probed previously with PEG-MAL.1,37

1384

Figure 5. Relative accessibility along the ribosomal exit tunnel. (a) Change in accessibility with number of amino acids from the PTC. The n-fold change in accessibility was calculated as the ratio of modification rate constants for MAL-ET modification of 62C or 73C engineered into separate tape measures designed to position the cysteine residues at the indicated distances from the PTC, normalized to the rate constant for modification of the corresponding cysteine outside the tunnel, located ≥67 residues from the PTC (see Materials and Methods). Corresponding tunnel distances (in Å) are indicated above the red arrows and are calculated using the all-extended tape measure.1 (b) Two models of the ribosomal tunnel. One indicates a 100 Å long tunnel that widens in the last 20 Å near the exit port (left). The other indicates that the tunnel is 80 Å long but is surrounded by chaperone proteins/factors at the exit (right). Both models predict a 20 Å region that is distinctly different from the cytosol, has a negative electrostatic potential, and may host initial tertiary folding events.33,47

Effect of charged amino acid in the nascent peptide Having defined the electrostatic potentials and relative accessibilities of the peptide in different locations of the tunnel, we could next address an equally important issue: what is the nature of the field when a charged side-chain is introduced into the tunnel during normal translation? How far from a charged side-chain (e.g. arginine) is the charge felt,

Ribosomal Tunnel Electrostatics

i.e. what is the effective length constant for the local electrostatic field generated by the charged sidechain? We do not know, for example, the consequence of introducing a charged side-chain into the nascent peptide, specifically whether the charge is distributed uniformly along the length of the tunnel. To address this issue, we performed an arginine scan of residues in 73CΔ27 in the vicinity of 73C, which is ∼ 80 Å from the PTC (–22 mV; Figure 4). We substituted an arginine downstream, either immediately adjacent to 73C (73CΔ27-R1), two away (73CΔ27-R2), or three away (73CΔ27-R3) from 73C, replacing residues P, Q, T, respectively (Figure 3(a)), and measured the modification rates of 73C by MALET and MAL-ES. All three mutants, 73CΔ27-R1, 73CΔ27-R2, and 73CΔ27-R3, increase the absolute modification rates (Table1). However, the ratio of the rates, ρcys, and hence the electrostatic potential, Ψ, only changes for 73CΔ27-R1 (–13.7( ± 1.0) mV) compared to 73CΔ27 itself (–21.4( ± 2.2) mV; Figure 6 and Table 1). The observed depolarization for 73CΔ27-R1 is expected. The lack of effect of an arginine located two or three residues away from 73C indicates that the charge falls off with distance, suggesting that the tunnel lumen has a dielectric that is high enough to screen the charge of the arginine side-chain. The precise distance dependence of the potential cannot be inferred, because we do not know the orientation of either the charged guanidium group or the cysteine side-chain. To confirm the finding that a positive charge adjacent to 73C depolarizes the potential at 73C, we substituted an oppositely charged aspartate in the same position (73CΔ27-D1) and two residues away (73CΔ27-D2). The adjacent substitution produced an equal and opposite effect to 73CΔ27-R1 on the electrostatic potential, hyperpolarizing the potential at 73C to − 29 mV. The more distant substitution produced no change in potential. Substitution of a neutral glutamine in the adjacent position (73CΔ27Q1) confirmed that the effects we measure are due to altered electrostatic potential. There was no difference in electrostatic potential for 73CΔ27 versus 73CΔ27-Q1. A change in accessibility, as manifest in the increased rates of both MAL-ET and MAL-ES (Table 1), was also detected when R or Q was substituted into the tape measure either adjacent to the cysteine or two or three residues away. We propose that this change in accessibility is due to either the length of the side-chain and/or to the nature of the side-chain (presence of an NH2 group), and not to the charge of the side-chain because Q substitution produces a similar increase in modification rates of both MAL reagents. Consistent with this hypothesis, the shorter side-chain of the D located two residues away from the cysteine (D2), a position that has no effect on electrostatic potential, also had no significant effect (p = 0.15, Student's t-test) on accessibility, i.e. on MAL-ET modification rate. At the adjacent position, both electrostatic and steric effects prevail and D1 substitution therefore results in an increase in the rate of MAL-ET, but not MAL-ES.

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Discussion Experimental approach

Figure 6. Effect of introducing a charge on the electrostatic potential in the tunnel. (a) Cartoon indicating the location of substituted arginine (blue), aspartate (red), and glutamine (white) in 73CΔ27. Cysteine 73C (black) is inside the tunnel, 27 residues from the PTC. An arginine introduced adjacent to 73C is labeled R1; an arginine introduced two or three residues away is labeled R2 and R3, respectively. Aspartate in the corresponding positions is labeled D1 and D2. Glutamine substitution adjacent to 73C is labeled Q1. (b) The nascent peptide was treated with either MAL-ET (10 μM) or MAL-ES (100 μM), pegylated, fractionated on gels, and analyzed to give modification rate constants. A ratio of rate constants was calculated as described in Materials and Methods to give the electrostatic potentials shown for each nascent peptide. The Ψ values for 73CΔ27 with a mutated residue at positions 1, 2, and 3 are indicated as means ± SEM (n = 3).

Our results clearly suggest a side-chain sensitive dynamic rearrangement of nascent peptide and/or tunnel. The tunnel walls may rearrange locally in response to a long side-chain (∼ 6 Å), and/or the peptide itself may alter its disposition and torsional angles in the tunnel to compensate for the cramped quarters. Although only the charge of the side-chain determines the local electrostatic potential, we propose that the length of the side-chain influences the accessibility and may affect dynamic conformations of the tunnel and/or the peptide.

To assess the validity of our estimates of tunnel electrostatics, we had to consider three general factors: the nature of the reagents, the access route, and the rate-limiting step in modification. First, although MTS reagents have been used to measure electrostatic potential,23–26,29 the assumption that they can uniquely estimate potential, without regard to properties like the size, shape, or chemistry of the reagents, has not been tested explicitly. Here, we demonstrate that two different sets of positively and negatively charged modifiers, despite their different chemistries, yield similar electrostatic potentials. Thiol reactions with MTS reagents involve a disulfide exchange, whereas MAL reagents produce a stable carbon-sulfur bond. Regarding the shape and size of the two sets of reagents, molecular modeling (ab initio optimization (HF/6-31++G*) of the structures using Gaussian03‡ Figure 1(a)) shows that for either pair of positively and negatively charged probes, the ratio of linear distances between the charged group and the reactive site is similar for both MTS and MAL reagents, and that the relative volumes of the reagents are similar (∼250 Å3). The molecular volume is slightly larger for the positive reagents, which would predict a slightly slower rate for the bulkier reagent. This is not observed. The positively charged reagent always has faster kinetics of modification. Another indication that a difference in reagent size between positively and negatively charged reagents is not a factor comes from the observation that both the narrowest and the widest part of the tunnel (see Figure 4) give the same potential, ∼− 19 mV. Moreover, evidence supporting our interpretation that charge pairs of MTS and MAL reagents uniquely estimate potential is that substitution of aspartate (D1) or arginine (R1) adjacent to 73C in 73CΔ27 shifts the potential approximately the same amount in opposite directions, whereas substitution of a glutamine (Q1) at this position does not alter the measured electrostatic potential. The second issue concerning the validity of our estimates is consideration of the access route to the modifiable tunnel cysteine residues. Small channels in the ribosome leading to the ribosomal exit tunnel are only large enough for water molecules and would not accommodate the larger MAL and MTS reagents.13 Thus, access to the tunnel lumen and nascent peptide cysteine residues occurs through the exit port and/or the PTC. The third issue concerns which of two consecutive steps is rate limiting: diffusion of the reagent to the cysteine or reaction of the reagent with the cysteine. Three lines of evidence support the idea that an

‡ http://www.gaussian.com

1386 equilibrium distribution of modifiers is achieved rapidly compared with the modification reaction. First, the kinetics of modification in all cases, both inside and outside the tunnel, are well-fit by a single exponential function and the rate depends linearly on reagent concentration. However, in all diffusion processes molecules move as the square root of time.38 Second, if diffusion were rate limiting, then all the reagents would have similar modification rate constants for a given cysteine. This is not observed, rather the rate constants differ by as much as 100-fold. Third, the results obtained with R1 and D1 mutants are incompatible with rate-limiting diffusion. These substitutions produce the expected equal and opposite effects for charged species on the electrostatic potential at 73C in 73CΔ27, which cannot be explained readily by an equal and opposite effect induced by the two different peptide side-chains on diffusion of the reagents. Electrostatics along the tunnel The tunnel maintains a robust electrostatic environment that modestly (but significantly, p b 0.001, ANOVA, Student-Newman-Keuls method) changes from the PTC to the exit port. The potentials range from approximately − 8 mV to − 22 mV. These electrostatic potentials are generated by permanent charges, partial charges, and the dipoles or quadrupoles lining the tunnel. The potentials are therefore dependent on rRNA, ribosomal proteins, ions, and the dielectric properties of the tunnel. One may speculate that the modest potentials are designed as a compromise to accommodate the wide range of amino acids (non-polar, polar, acidic, and basic) that comprise natural peptides traversing the tunnel. The least negative potential is in the neighborhood of the PTC, a region reported to contain a relatively high Mg2+ content.39 The regions of most negative potential, ∼− 20 mV, are in the vicinity of the narrowest part of the tunnel, into which ribosomal proteins L4 and L22 (L17 in mammalian ribosomes) protrude, and in the last 20 Å of the tunnel. In the former case, the proteins themselves may contribute to the increased negativity and/or the electrostatic field may be focused to enhance the negative potential. 17,18 In the latter case, the increased negativity could arise from the welcoming committee of chaperones grouped at the exit port. It is intriguing to note that a nascent peptide cysteine located outside but adjacent to the exit port has a similar accessibility (Figure 5), but a very different electrostatic potential from the same cysteine located out in free solution some distance away from the tunnel (Figure 4(a) and Table 1; compare 62CΔ74, − 4.9 mV and 62CΔ34, − 18.7 mV). We suggest that the welcoming committee does not hinder access to small molecules, but its electrostatic potential exerts an influence on the emerging peptide. The magnitudes of the tunnel potentials are consistent with values for protein crevices and vestibules measured using a ratio of rate constants

Ribosomal Tunnel Electrostatics

of MTS modification of engineered cysteine residues. In voltage-gated sodium channels, potentials can range from − 14 mV to − 45 mV25 in the gating pore, and from − 12 mV to +14 mV in the vicinity of the S3 voltage-sensor.26 In chloride channels, electrostatic potentials in the inner pore region range from 0 to + 49 mV.24 However, in the permeation pore of the acetylcholine receptor, potentials exist over an extremely wide range,23 likely due to the rings of negatively charged residues that point into the pore. Regarding the introduction of a charged sidechain in proximity to a modifiable cysteine residue in the tunnel, 73C, we can speculate why a charged residue located two or three residues away from 73 does not alter the electrostatic potential at residue 73C. R2 and D2 are farther away than R1, at a distance larger than the effective Debye length within this region of the tunnel. Unfortunately, we cannot know the exact distance between the introduced charged moieties and the thiol of the modifiable cysteine, because the orientations of the side-chains of the residues are unknown. For example, the thiol of 73C and the guanidinium group of R2 could be as far apart as 16 Å. Whatever the actual distance, the dielectric constant in the tunnel is evidently high enough for water and dissolved ions to screen the introduced charge. This is consistent with the different electrostatic potentials along the tunnel (Figure 4; Table 1). The effective dielectric constant depends critically on the size and shape of an aqueous crevice.16 If, for example, introduction of a residue causes a local increase in the volume of the tunnel, this would increase the ability of mobile ions and water to screen electrostatic potentials near the target cysteine, increasing the dielectric constant and damping the projection of electrostatic potentials along the tunnel. Nonetheless, the fact that a charge significantly alters the local electrostatic potential raises the exciting possibility that the electrostatic potentials in the tunnel are dynamic and tuned by the presence of an elongating nascent peptide. As a peptide passes through regions of different electrostatic potential (Figure 4), it will sequentially depolarize and hyperpolarize the local electrostatic potential in accord with its primary sequence that specifies the number and distribution of charged side-chains. This predicts a wave of electrostatic potentials in the tunnel during translation of each unique peptide. In addition to the effect of charged side-chains on the nearby electrostatic potential, another intriguing possibility arises from the measured rate constants. The modification rate constants in the arginine-scan experiments increase even when the electrostatic potentials are unchanged (Table 1). This suggests that the accessibility increases (see Materials and Methods and Elinder et al.27), i.e. the space around the cysteine widens. Why does it widen? Either because the tunnel wall moves (is dynamic) or the peptide reorients in the tunnel and this reorientation is dependent on the side-chain.

Ribosomal Tunnel Electrostatics

Possible role of tunnel electrostatics Intrinsic electrostatic potentials govern many biological interactions, including enzyme–substrate, protein–protein, and protein–nucleic acid interactions. 40 It is likely that electrostatic potentials similarly govern interactions of a nascent peptide with the ribosome, attendant chaperones, and factors involved in translation. An electric field can reorient permanent dipoles and redistribute charges, e.g. mobile ions. These responses can have large effects on macromolecular folding, conformational stability, association and dissociation rates, and catalysis.28 Tunnel electrostatics may be considered from two points of view. The first consideration is that the overall negative potential will have consequences for the ionization of groups that are near their pKa value in the tunnel, e.g. histidine side-chains and the N terminus of the nascent peptide. It will also change ion concentrations in the tunnel. The second consideration is that the variation of electrical potential in the tunnel may have consequences for folding of the nascent peptide. Interactions of the backbone with the tunnel wall will be dependent on the local electrostatic potential gradient, i.e. the electric field. As the backbone organizes itself, helix dipoles might have long-range interactions with the local potential. For example, the more negative electrostatic potential near the exit would favor the N terminus forming a helix, since this will be at the positive end of the helix dipole. Indeed, helix formation of native transmembrane segments of the voltage-gated potassium channel, Kv1.3, occurs preferentially in the last 20 Å of the tunnel,1,41 where the electrostatic potential is ∼− 20 mV and the tunnel widens (see below and Figure 5). Could the magnitudes (− 8 mV to − 22 mV) of the electrostatic potentials be significant? Consider two examples. First, what is the effect of a − 22 mV electrostatic potential on ion concentrations at a particular location in the tunnel? This potential results in a ∼ 1 kcal/mol enhancement of the free energy for attracting a Mg2+ to this location, which could produce a ∼ 6-fold increase in Mg2+ concentration. Moreover, a hydrated Mg2+ is ∼ 8 Å in diameter, perhaps too bulky for small ribosomal crevices. A negative tunnel can reduce the energetic cost to dehydrate Mg2+ , making Mg2+ binding in the tunnel more favorable. Second, we do not know how many charged residues on the nascent peptide are involved in a folding reaction, i.e. the steepness of any voltage-dependent folding. The more charges, the greater the sensitivity of the equilibrium (or transition state, i.e. rate) in the folding reaction. In a simplistic model in which exactly one electronic charge traverses the local electric field during a folding reaction, a ∼ 2-fold change in equilibrium or transition state energy would be predicted for a change of 22 mV. If more than one charge moves through the local electric field during folding, this conformational change will be more sensitive to the electric field.

1387 Finally, the electrostatic potential in the tunnel may modulate translation rates and/or energetics of chaperone interactions at the exit port, and consequently the ease and speed of folding. For example, several arrest sequences contain charged residues that are critical for pausing events,2,19–22 and charge changes of amino acids in ribosomal protein L22 that point into the tunnel alter arrest.2,42 Moreover, insertion of a polylysine tract into a nascent peptide causes co-translational arrest and degradation of the peptide.43 All of these observations implicate modulation of translation events by local electrostatic potentials. The shape of the tunnel The modification rate constants obtained by using MAL-ET may also inform us about the shape of the tunnel. A change in accessibility may be calculated from the rate constants for each cysteine in the tape measure relative to a cysteine outside of the tunnel (Figure 5; see Materials and Methods and Elinder et al. 27 ). Using the tape measure, we have now determined the relative accessibility along the tunnel. This is an operational definition of the functional ribosomal tunnel. Previously, we used PEG-MAL, which is considerably larger than the small MAL-ET and MAL-ES reagents used here, to measure accessibility as determined by distancedependent final extent of labeling. In those studies, we measured the accessibility of the last 20 Å of the tunnel.1 Regardless of small versus large maleimides, or kinetics versus final extent of labeling, the same change in accessibility with distance was obtained. Two models (Figure 5(b)) are consistent with our map of accessibility. Model (a) proposes that the tunnel wall extends a distance of 100 Å but flares near the exit port. Model (b) proposes that the tunnel is only 80 Å in length and that chaperone proteins extend the effective length of the tunnel to produce a functional length of 100 Å. Both models are consistent with the measured accessibility and electrostatic potentials in this region. Regardless of the shape of the tunnel and its determinants, we may conclude that the tunnel– peptide complex is dynamic and sensitive to the length, and/or perhaps the nature, of the peptide side-chains. The increased accessibility for the R and Q-substituted tape measures suggests that there are shape changes in the tunnel–peptide complex. This is consistent with macrolide-induced conformational rearrangement of a tunnel wall component3 and nascent peptide-induced increase in the internal volume of the exit tunnel.44

Materials and Methods Constructs and in vitro translation Standard methods of bacterial transformation, plasmid DNA preparation and restriction enzyme analysis were used. The nucleotide sequences of all mutants were

1388 confirmed by automated-cycle sequencing performed by the DNA Sequencing Facility at the University of Pennsylvania School of Medicine on an ABI 377 sequencer using Big dye terminator chemistry (ABI). The tape measure DNAs were sequenced throughout the entire coding region. Engineered cysteine residues were introduced into pSP/Kv1.3/cysteine-free45 using Stratagene's QuikChange site-directed mutagenesis kit. In all experiments, we used a molecular tape measure, which is the C-terminal 44 amino acids of the first 95 amino acids of the T1 domain of Kv1.3.1 Five native cysteine residues in the tape measure constructs, including Cys71, were replaced with serine residues to give a cysteine-free background. Two α-helices, α1 (from Leu67 to Leu70) and α2 (from Pro81 to Arg83) in the wild-type T1 domain, were deleted and a new BstEII restriction site was engineered at Arg101 using Stratagene's QuikChange site-directed mutagenesis kit. This new site inserted a serine between Arg101 and the PTC. When a BstEII-cut tape measure construct is translated, it generates a nascent peptide of 95 amino acids, which migrates at ∼ 15 kDa on NuPAGE gels.1 In most of the experiments, the tape measure was positioned to span the length of the ribosomal exit tunnel using the BstEII restriction enzyme. To determine tunnel electrostatics, two separate, mutated tape measure constructs were made: R62C (E64Q) and F73C (E75Q) tape measures. The second mutation in parenthesis was designed to eliminate charged side-chains that might complicate determination of rate constants for modification of the nearby cysteine. These constructs are referred to as 62CΔ34 and 73Δ27, respectively. To move 62C closer to the PTC site, 4, 7, 14, and 21 residues were deleted from the C terminus of the 62CΔ34 tape measure constructs using PCR methods to generate 62CΔ30, 62CΔ27, 62CΔ20, and 62CΔ13 tape measures. To move 73C closer to the PTC site, 7, 14, and 21 residues were deleted from the C terminus of the 73CΔ27 tape measure constructs to generate 73CΔ20, 73CΔ13, and 73CΔ6 tape measures. All these constructs shared a common 5′ end primer: 5′-AgA ggA TcT ggc TAg c(NheI site)gA Tg- 3′, which was situated about 250 bp upstream from the starting codon, whereas the 3′ end primers were designed appropriately upstream of the 3′ end of the construct to yield the shortened tape measures. All PCR fragments were purified and sequenced for the entire coding region and used as templates for transcription. To deliberately introduce a charged side-chain or a neutral control, R1/D1/Q1, R2/D2, or R3 were introduced into the 73CΔ27 tape measure by mutating residues P74, Q75, or T76, respectively, with Stratagene's QuikChange site-directed mutagenesis kit. The following additional control constructs were made. A natural NcoI-digestion site at position 141/142 was used to generate nascent peptides in which R62C and F73C were placed 74 and 67 residues , respectively, away from the PTC. These constructs are referred to as 62CΔ74 and 73CΔ67, respectively. Mutation Q72C was introduced into the NcoI-cut tape measure using Stratagene's QuikChange site-directed mutagenesis kit. Capped complementary RNA was synthesized in vitro from linearized templates using Sp6 RNA polymerase (Promega). Linearized templates for Kv1.3 biogenic intermediates were generated using BstEII enzyme. Proteins were translated in vitro with [35S]methionine Express (2 μl per 25 μl translation mixture; ∼ 10 μCi μl− 1; DuPont NEN Research Products) for 1 h at 22 °C in rabbit reticulocyte lysate according to the Promega Protocol and Application Guide.

Ribosomal Tunnel Electrostatics Synthesis and characterization of maleimide reagents Sulfo (MAL-ES) and trimethylammonium N-ethyl (MAL-ET) maleimides were synthesized as described.46 Briefly, taurine or 2-aminoethyltrimethylammonium (1 mmol) was dissolved in 1 ml of saturated aqueous sodium bicarbonate, the solution was chilled to 0 °C, and N-methylcarbonyl maleimide (1 mmol) was added. After stirring for 1 h, the ice bath was removed and the reaction was allowed to warm to room temperature (1 h). The reaction was neutralized to pH 4 – 5 with aqueous 1% (v/v) H2SO4 and the solvents were removed by lyophilization. The crude material was dissolved in water with 0.1% (w/v) trifluoroacetic acid (TFA) and passed down a low pressure C18 column (10 ml) eluting with 0.1% TFA. The fractions containing product were subsequently purified by C18 reversed phase HPLC (10 mm × 250 mm column) using an isocratic elution (0.1% TFA in water; 5 ml/min). Collected fractions were pooled and the solvents were removed by lyophilization. Yields ranged from 40%–75%. MAL-ES (Na+): 1H NMR (400 MHz, 2H2O) δ 3.17 (t, 2 H, J = 6.6), 3.92 (t, 2 H, J = 6.6), 6.85 (s, 2 H); 13C NMR (internal standard: acetonitrile) δ 33.78, 48.32, 135.1, 172.9; HRMS (type) calculated for C6H6O5NS (M)– 203.9967, found 203.9994. MAL-ET (TFA–): 1H NMR (400 MHz, methanol-d4) δ 3.22 (s, 9 H), 3.60 (t, 2 H, J = 6.7), 4.00 (t, 2 H, J = 6.7), 6.93 (s, 2 H); 13C NMR δ 32.79, 54.06 (t), 64.49 (t), 136.1, 172.0; HRMS (type) calculated for C9H15O2N2 (M)+ 183.1134, found 183.1130. Kinetics of cysteine modification with MTS or maleimides As described,1 translation product (5–10 μl for peptides containing two or more methionine residues and 10–15 μl for peptides containing a single methionine) was centrifuged through a sucrose cushion (120 μl; 0.5 M sucrose, 100 mM KCl, 5 mM MgCl2, 50 mM Hepes (pH 7.3), no added DTT) for 20 min at 70,000 rpm with a TLA 100.3 Beckman ultra-centrifuge rotor at 4 °C to isolate ribosome-bound peptide. The supernatant was completely removed (critical requirement) and the pellet was resuspended on ice in 100– 500 μl of buffer containing 100 mM NaCl, 2.5–5 mM Mg2+, 20 mM Hepes (pH 7.3). Effective resuspension required careful (avoiding bubble formation) and repetitive (N 100 times) pipetting. When using maleimide reagents, we kept small aliquots of lyophilized MAL-ES and MAL-ET at −80 °C and immediately before using, we serially diluted separately a (+/−) pair of MAL reagents with the same buffer described above to achieve a final working stock concentration of 10 times their final working concentration (a range of 3 μM–10 μM for MAL-ET and 30 μM–100 μM for MALES). The reaction was started by adding 1/10 dilution of the MAL reagent to the peptide-containing buffer. The reaction mixture was incubated on ice for times indicated in the time course experiments, quenched by addition of 2 mM DTT, and centrifuged through a sucrose cushion (120 μl; 0.5 M sucrose, 100 mM KCl, 5 mM MgCl2, 50 mM Hepes, 1 mM DTT (pH 7.3)). The pellet was resuspended by pipetting in 25 μl buffer containing 20 mM Hepes, 1%(w/v) SDS, 10 μg ml− 1 of RNase, 100 mM NaCl, and 50 μM DTT. When using MTS reagents, we made a working stock of 100 mM MTS-ES or MTS-ET in distilled, deionized water, which

Ribosomal Tunnel Electrostatics

1389

was stored at −80 °C. The reaction protocol was similar to that used for MAL-reagent reactions except that cysteine (1–2 mM) was used instead of DTT as a quenching/reducing agent. Pegylation was started by adding a final concentration of 2 mM PEG-MAL and continued for 3 h at 4∼8 °C in a refrigerator. The reaction was then terminated by adding 50 mM DTT and vortexing. For each time course experiment, the reaction was sampled at five time points between 0 and ∼2 h. Time course data were fit to a single-exponential decay function to calculate the rate constants. The electrostatic potential in the vicinity of an engineered cysteine was calculated from the formulation by Karlin and coworkers.23,29 This entails calculating a “ratio-of-ratios” to estimate the relative reactivity of the cysteine to positively charged and negatively charged reagents compared to the relative reactivity of a free thiol in solution, e.g. β-mercaptoethanol. The relative reactivity of β-mercaptoethanol to MAL reagents, under identical conditions used for modification of a cysteine in the nascent peptide, was determined from stopped-flow experiments using a PbP Spectrakinetic Monochromator 05–109 (Applied Photophysics). The decrease in absorbance at 236 nm indicates reaction of the maleimide. Control experiments were performed using two-reservoir syringes filled with buffer alone, MAL probes alone, βmercaptoethanol alone. These controls showed no change in absorbance upon mixture of the contents of both syringes in the optical chamber. The reaction of βmercaptoethanol (100 μM) with MAL-ET or MAL-ES (1 mM), carried out at 6.7 °C, was manifest as a timedependent decrease in absorbance, which was fit with a single-exponential function to give modification rate constants of 1216 M− 1s− 1 and 174 M− 1s− 1, respectively, for MAL-ET (n = 3) and MAL-ES (n = 3). The ratio MALET to MAL-ES is 6.99. The relative reactivity for MTS reagents was approximated to be 7.0 based on temperature dependence and reported values29 (but see Karlin and Akabas30) The ratio-of-ratios, ρR, is equal to the ratio of measured modification rate constants divided by 6.99. The relationship between relative reactivity of the cysteine and the electrostatic potential, Ψ, is ρR = exp ((–zET + zES)FΨ/RT), zET and zES are the electronic charges of the reagents, equal to + 1 and − 1, respectively.23 Rearrangement of this equation gives the electrostatic potential at a given cysteine in the peptide, Ψ = -(RT/2F )(lnρR) = –12.5 lnρR. Data analysis The following analysis is taken from Elinder et al.27 for Shaker K+ channel movement of the S4 segment in response to voltage. The modification rate, ρ, of a given cysteine in the nascent peptide is equal to: U ¼ kMAL Acys PS 2½MAL

ð2Þ

where kMAL is the intrinsic reaction rate of a cysteine for a specific reagent, Acys is the accessibility of the thiol group, PS- is the probability of the thiol group being in its unprotonated thiolate state, and [MAL] is the local concentration of MAL-ET in the vicinity of the cysteine. PS- depends on the local electrostatic potential (Ψ). Experimentally determined rate constants presented in the Results and Table 1 are equal to the product (kMAL Acys PS-). A negative Ψ attracts protons to the thiol, a positive

Ψ repels protons (i.e. effectively raises the local pH). This is expressed quantitatively as: PS  ¼ 1=ð1 þ 10ðpKapHÞ expðCF=RTÞÞ

ð3Þ

where pKa is the pH at which 50% of the cysteines are ionized (8.5 in physiological salt solution) and pH is the negative logarithm of the [H+] in the bulk solution. If the surface potential inside the tunnel is negative (see Results for 62C and 73C scan of the tunnel at different positions in the tunnel, i.e. different chain lengths), then pHlocal≪pKa and equation (3) reduces to: PS  ¼ 1=ð10ðpKapHÞ expðCF=RTÞÞ

ð4Þ

As with protons, the local concentration of charged maleimide is also exponentially dependent on the local electrostatic potential: ½MAL ¼ ½MALbulk expðzCF=RTÞ

ð5Þ

where z is the valence of the charged MAL reagent. Substituting equations (4) and (5) into equation (2), and making a ratio of the modification rates for a given cysteine measured in two different constructs (ρ1/ρ2, e.g. presence and absence of an arginine in an adjacent position (73CΔ27-R1)/(73CΔ27)), the following relationship is derived: U1 =U2 ¼ fkMAL Acys,1 10ðpKapHÞ expðC2 F=RTÞ½MALbulk  expðz C1 F=RTÞg=fkMAL Acys;2 10ðpKapHÞ  expðC1 F=RTÞ½MALbulk expðz C2 F=RTÞg ð6Þ If the charge on the maleimide is +1 (MAL-ET), then equation (6) simplifies to: U1 =U2 ¼ ðAcys;1 =Acys;2 Þ

ð7Þ

indicating that the relative rates of modification of the cysteine by the positively charged reagent equals the fold increase in accessibility for the cysteine in nascent peptide 1 versus nascent peptide 2. If the charge on the maleimide is − 1 (MAL-ES), then equation (6) reduces to: U1 =U2 ¼ ðAcys;1 =Acys;2 Þðexpð2FðC1  C2 Þ=RTÞÞ

ð8Þ

Because hydrolysis of MTS-ET is rapid enough to change the concentration of the reagent during the modification reaction and in the initial preparation of stock solutions, we measured the hydrolysis rate under the conditions used in the modification experiments. The rate constant for hydrolysis, khyd, is 14.8 × 10− 4 s− 1 at pH 7.3, 0–4 °C. This is in good agreement with the rate constants measured by Karlin and Akabas (10 × 10− 4 s− 1 −12 × 10− 4 s− 1 at pH 7.0, 20 °C). The value of khyd was used in equation (9) (taken from Nguyen and Horn26) to fit the MTS-ET modification data to obtain a modification rate constant, kmod: Smod ðtÞ ¼ 1  exp½ðkmod =khyd ÞMo ð1  expðtkhydÞÞ ð9Þ where Smod(t) is the irreversible modification and Mo is the initial concentration of MTS-ET. Gel electrophoresis and fluorography All final samples were heated at 70 °C for 10 min in 1 × of NUPAGE loading buffer before loading onto the gel.

1390 Electrophoresis was performed using the NuPAGE system and precast bis–Tris 10% or 12% (w/v) gels and Mes running buffer. Gels were soaked in Amplify (Amersham) to enhance 35S fluorography, dried and exposed to Kodak X-AR film at −70 °C. Typical exposure times were 16–30 h. Quantification of gels was carried out directly using a Molecular Dynamics PhosphorImager.

Acknowledgements We thank Dr M. Ostap for assistance with the stopped-flow experiments and use of his equipment, Drs M. Gunner and K. Giangiacomo for helpful discussion, and Drs R. Horn and J. Lear for critical reading of the manuscript. This work was supported by National Institutes of Health grant GM 52302.

References 1. Lu, J. & Deutsch, C. (2005). Secondary structure formation of a transmembrane segment in Kv channels. Biochemistry, 44, 8230–8243. 2. Nakatogawa, H. & Ito, K. (2002). The ribosomal exit tunnel functions as a discriminating gate. Cell, 108, 629–636. 3. Berisio, R., Schluenzen, F., Harms, J., Bashan, A., Auerbach, T., Baram, D. & Yonath, A. (2003). Structural insight into the role of the ribosomal tunnel in cellular regulation. Nature Struct. Biol. 10, 366–370. 4. Woolhead, C. A., McCormick, P. J. & Johnson, A. E. (2004). Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell, 116, 725–736. 5. Woolhead, C. A., Johnson, A. E. & Bernstein, H. D. (2006). Translation arrest requires two-way communication between a nascent polypeptide and the ribosome. Mol. Cell, 22, 587–598. 6. Liao, S., Lin, J., Do, H. & Johnson, A. E. (1997). Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration. Cell, 90, 31–41. 7. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000). The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science, 289, 905–920. 8. Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. (2000). The structural basis of ribosome activity in peptide bond synthesis. Science, 289, 920–930. 9. Menetret, J. F., Neuhof, A., Morgan, D. G., Plath, K., Radermacher, M., Rapoport, T. A. & Akey, C. W. (2000). The structure of ribosome-channel complexes engaged in protein translocation. Mol. Cell, 6, 1219–1232. 10. Beckmann, R., Spahn, C. M., Eswar, N., Helmers, J., Penczek, P. A., Sali, A. et al. (2001). Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell, 107, 361–372. 11. Crowley, K. S., Reinhart, G. D. & Johnson, A. E. (1993). The signal sequence moves through a ribosomal tunnel into a non-cytoplasmic aqueous environment at the ER membrane early in translocation. Cell, 73, 1101–1115.

Ribosomal Tunnel Electrostatics 12. Hamman, B. D., Chen, J. C., Johnson, E. E. & Johnson, A. E. (1997). The aqueous pore through the translocon has a diameter of 40–60 A during cotranslational protein translocation at the ER membrane. Cell, 89, 535–544. 13. Voss, N. R., Gerstein, M., Steitz, T. A. & Moore, P. B. (2006). The geometry of the ribosomal polypeptide exit tunnel. J. Mol. Biol. 360, 893–906. 14. Ziv, G., Haran, G. & Thirumalai, D. (2005). Ribosome exit tunnel can entropically stabilize alpha-helices. Proc. Natl Acad. Sci. USA, 102, 18956–18961. 15. Snir, Y. & Kamien, R. D. (2005). Entropically driven helix formation. Science, 307, 1067. 16. Sansom, M. S., Smith, G. R., Adcock, C. & Biggin, P. C. (1997). The dielectric properties of water within model transbilayer pores. Biophys. J. 73, 2404–2415. 17. Getzoff, E. D., Tainer, J. A., Weiner, P. K., Kollman, P. A., Richardson, J. S. & Richardson, D. C. (1983). Electrostatic recognition between superoxide and copper, zinc superoxide dismutase. Nature, 306, 287–290. 18. Klapper, I., Hagstrom, R., Fine, R., Sharp, K. & Honig, B. (1986). Focusing of electric fields in the active site of Cu-Zn superoxide dismutase: effects of ionic strength and amino-acid modification. Proteins: Struct. Funct. Genet. 1, 47–59. 19. Lovett, P. S. & Rogers, E. J. (1996). Ribosome regulation by the nascent peptide. Microbiol. Rev. 60, 366–385. 20. Fang, P., Wang, Z. & Sachs, M. S. (2000). Evolutionarily conserved features of the arginine attenuator peptide provide the necessary requirements for its function in translational regulation. J. Biol. Chem. 275, 26710–26719. 21. Gong, F. & Yanofsky, C. (2001). Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyltRNA. J. Biol. Chem. 276, 1974–1983. 22. Tenson, T. & Ehrenberg, M. (2002). Regulatory nascent peptides in the ribosomal tunnel. Cell, 108, 591–594. 23. Pascual, J. M. & Karlin, A. (1998). State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit. J. Gen. Physiol. 111, 717–739. 24. Lin, C. W. & Chen, T. Y. (2003). Probing the pore of ClC-0 by substituted cysteine accessibility method using methane thiosulfonate reagents. J. Gen. Physiol. 122, 147–159. 25. Yang, N., George, A. L. J. & Horn, R. (1997). Probing the outer vestibule of a sodium channel voltage sensor. Biophys. J. 73, 2260–2268. 26. Nguyen, T. P. & Horn, R. (2002). Movement and crevices around a sodium channel S3 segment. J. Gen. Physiol. 120, 419–436. 27. Elinder, F., Mannikko, R. & Larsson, H. P. (2001). S4 charges move close to residues in the pore domain during activation in a K channel. J. Gen. Physiol. 118, 1–10. 28. Sharp, K. A. & Honig, B. (1990). Electrostatic interactions in macromolecules: theory and applications. Annu. Rev. Biophys. Biophys. Chem. 19, 301–332. 29. Stauffer, D. A. & Karlin, A. (1994). Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. Biochemistry, 33, 6840–6849.

Ribosomal Tunnel Electrostatics 30. Karlin, A. & Akabas, M. H. (1998). Substituted-cysteine accessibility method. Methods Enzymol. 293, 123–145. 31. Lu, J. & Deutsch, C. (2001). Pegylation: a method for assessing topological accessibilities in Kv1.3. Biochemistry, 40, 13288–13301. 32. Robinson, J. M. & Deutsch, C. (2005). Coupled tertiary folding and oligomerization of the T1 domain of Kv channels. Neuron, 45, 223–232. 33. Kosolapov, A., Tu, L., Wang, J. & Deutsch, C. (2004). Structure acquisition of the T1 domain of Kv1.3 during biogenesis. Neuron, 44, 295–307. 34. Kreusch, A., Pfaffinger, P. J., Stevens, C. F. & Choe, S. (1998). Crystal structure of the tetramerization domain of the Shaker potassium channel. Nature, 392, 945–948. 35. Minor, D. L., Lin, Y. F., Mobley, B. C., Avelar, A., Jan, Y. N., Jan, L. Y. & Berger, J. M. (2000). The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel. Cell, 102, 657–670. 36. Long, S. B., Campbell, E. B. & MacKinnon, R. (2005). Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science, 309, 897–903. 37. Lu, J. & Deutsch, C. (2005). Folding zones inside the ribosomal exit tunnel. Nature Struct. Mol. Biol. 12, 1123–1129. 38. Moore, W. J. (1972). Physical Chemistry, Prentice-Hall, Englewood Cliffs, NJ. 39. Klein, D. J., Moore, P. B. & Steitz, T. A. (2004). The contribution of metal ions to the structural stability of the large ribosomal subunit. RNA, 10, 1366–1379.

1391 40. Honig, B. & Nicholls, A. (1995). Classical electrostatics in biology and chemistry. Science, 268, 1144–1149. 41. Tu, L., Wang, J. & Deutsch, C. (2007). Biogenesis of the T1-S1 linker of voltage-gated K+ channels. Biochemistry, 46, 8075–8084. 42. Cruz-Vera, L. R., Rajagopal, S., Squires, C. & Yanofsky, C. (2005). Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression. Mol. Cell, 19, 333–343. 43. Ito-Harashima, S., Kuroha, K., Tatematsu, T. & Inada, T. (2007). Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast. Genes Dev. 21, 519–524. 44. Gilbert, R. J., Fucini, P., Connell, S., Fuller, S. D., Nierhaus, K. H., Robinson, C. V. et al. (2004). Threedimensional structures of translating ribosomes by Cryo-EM. Mol. Cell, 14, 57–66. 45. Lu, J., Robinson, J. M., Edwards, D. & Deutsch, C. (2001). T1-T1 interactions occur in ER membranes while nascent Kv peptides are still attached to ribosomes. Biochemistry, 40, 10934–10946. 46. Sun, C., Aspland, S. E., Ballatore, C., Castillo, R., Smith, A. B. & Castellino, A. J. (2006). The design, synthesis, and evaluation of two universal doxorubicin-linkers: preparation of conjugates that retain topoisomerase II activity. Bioorg. Med. Chem. Letters, 16, 104–107. 47. Kosolapov, A. & Deutsch, C. (2006). Folding of T1 subdomains of nascent Kv1.3. FASEB J. 20, A965.

Edited by D. E. Draper (Received 27 April 2007; received in revised form 11 June 2007; accepted 12 June 2007) Available online 19 June 2007