Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23

The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23.     

基于时间分辨方法的LicT蛋白荧光动力学特性

使用时间分辨荧光方法,结合紫外吸收光谱和稳态荧光光谱技术,测量了LicT蛋白中色氨酸残基的荧光动力学特性,进而对LicT蛋白质激活前后的局部微环境和结构变化进行了研究。LicT蛋白质的激活态使得有关糖类利用的基因转录过程继续进行,促进机体新陈代谢。通过色氨酸残基的荧光发射和寿命的差异判断出激活型蛋白AC 141和野生型蛋白Q 22不同的结构性质和微环境差异。在此基础上,通过衰减相关光谱(DAS)和时间分辨发射光谱(TRES)阐释了两种蛋白色氨酸残基和溶剂的相互作用,说明了激活型AC 141的比野生型Q 22的结构更加紧密。此外,TRES还说明了蛋白中的色氨酸残基存在连续光谱弛豫过程。各向异性结果则对残基和整个蛋白的构象运动进行了阐述,说明了色氨酸残基在蛋白质体系内有独立的局部运动,且在激活型蛋白中该运动更加强烈。     

Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate

An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.     

Photophysical properties of ricin

The molar absorption coefficient of ricin in phosphate-buffered saline (PBS) at 279 nm was measured as (93,900+/-3300) L mol(-1) cm(-1). The concentration of ricin was determined using amino acid analysis. The absorption spectrum of ricin was interpreted in terms of 69% contribution from absorption by tryptophan residues and 31% contribution from absorption by tyrosine residues. The total dipole strength of the ricin band at 280 nm was determined to be (147+/-8) D2 and was consistent with the combined dipole strengths of 10 tryptophan ([11.7+/-1.0] D2) and 23 tyrosine ([1.4+/-0.2] D2) residues. The structure of ricin was used to determine the coupling of the tryptophan residues in ricin. The maximum interaction energy was found to be 424 cm(-1)/epsilon while the average interaction between any two pairs of tryptophan residues was approximately 18 cm(-1)/epsilon. In this study, epsilon is the dielectric constant inside the protein. The fluorescence from ricin, excited at 280 nm, was dominated by fluorescence from tryptophan residues suggesting the presence of energy transfer from tyrosine to tryptophan residues. The absorbance and fluorescence of ricin increased slightly when ricin was denatured in a high concentration of guanidine. Irreversible thermal unfolding of ricin occurred between 65 degrees C and 70 degrees C. (D=3.3364*10(-30) Cm, not SI unit, convenient unit for the magnitude of the electric dipole moment of molecules.).     

Porphyrin fluorescence dominates UV photoemission of folded cytochrome c

In this article we reinvestigate the bimodal fluorescence of cytochrome c (Cyt c) by using excitation-wavelength-dependent fluorescence spectroscopy. We show that its major contributions at pH 3-7 do not arise from tryptophan (Trp-59) fluorescence as hitherto assumed. Instead, different chromophores of Cyt c contribute at different pH values. At pH 3-7, the porphyrin system contributes about 80% and tryptophan about 20% to the total fluorescence upon excitation of Cyt c at 280 nm. At pH 2, the fluorescence originates nearly completely from the tryptophan residue. Porphyrin fluorescence is still present at pH 2 but its contribution is too small for quantitative deconvolution. Our results show that the UV fluorescence of Cyt c has to be deconvoluted before it can be used to perform time-resolved measurements of the folding of this small protein.     

Probing folded and unfolded states of outer membrane protein a with steady-state and time-resolved tryptophan fluorescence

Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions.     

Fluorescence quenching and time-resolved fluorescence studies on Trichosanthes dioica seed lectin

Fluorescence quenching and time-resolved fluorescence studies have been carried out on the Trichosanthes dioica seed lectin (TDSL). The emission lambdamax of native TDSL, seen at 328nm, shifts to 343nm upon denaturation with 6M guanidinium chloride. Quenching titrations were performed with neutral (acrylamide and succinimide) and ionic (I(-) and Cs(+)) quenchers in order to probe the exposure and accessibility of tryptophan residues of the protein. Maximum quenching was observed with acrylamide, followed by succinimide, iodide and Cs(+). Dramatic increase in the extent of quenching and other quenching parameters by all the quenchers were observed upon denaturation of TDSL, suggesting that all the tryptophan residues in native TDSL are buried in the hydrophobic core of the protein. Increase in the extent of quenching upon denaturation of TDSL was maximum with I(-) and minimum with Cs(+), suggesting the presence of positively charged residue(s), near at least one tryptophan residue. Addition of saccharide ligands such as methyl-beta-d-galactopyranoside and lactose led to a small, but reproducible decrease in the fluorescence intensity of the lectin. The presence of lactose provided a partial protection against quenching by I(-), Cs(+) and succinimide, but not acrylamide. In time-resolved fluorescence measurements the fluorescence decay curves could be best fitted to biexponential patterns with lifetimes of 4.09 and 1.53ns for native lectin, 3.40 and 1.65ns for the lectin in presence of 0.1M lactose and 3.50 and 1.40ns for denatured lectin.     

Fluorescence of cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid: a single tryptophan chi(1) rotamer model

A constrained derivative, cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid, cis-W3, was designed to test the rotamer model of tryptophan photophysics. The conformational constraint enforces a single chi(1) conformation, analogous to the chi(1) = 60 degrees rotamer of tryptophan. The side-chain torsion angles in the X-ray structure of cis-W3 were chi(1) = 58.5 degrees and chi(2) = -88.7 degrees. Molecular mechanics calculations suggested two chi(2) rotamers for cis-W3 in solution, -100 degrees and 80 degrees, analogous to the chi(2) = +/-90 degrees rotamers of tryptophan. The fluorescence decay of the cis-W3 zwitterion was biexponential with lifetimes of 3.1 and 0.3 ns at 25 degrees C. The relative amplitudes of the lifetime components match the chi(2) rotamer populations predicted by molecular mechanics. The longer lifetime represents the major chi(2) = -100 degrees rotamer. The shorter lifetime represents the minor chi(2) = 80 degrees rotamer having the ammonium group closer to C4 of the indole ring (labeled C5 in the cis-W3 X-ray structure). Intramolecular excited-state proton transfer occurs at indole C4 in the tryptophan zwitterion (Saito, I.; Sugiyama, H.; Yamamoto, A.; Muramatsu, S.; Matsuura,T. J. Am. Chem. Soc. 1984, 106, 4286-4287). Photochemical isotope exchange experiments showed that H-D exchange occurs exclusively at C5 in the cis-W3 zwitterion, consistent with the presence of the chi(2) = 80 degrees rotamer in solution. The rates of two nonradiative processes, excited-state proton and electron transfer, were measured for individual chi(2) rotamers. The excited-state proton-transfer rate was determined from H-D exchange and fluorescence lifetime data. The excited-state electron-transfer rate was determined from the temperature dependence of the fluorescence lifetime. The major quenching process in the -100 degrees rotamer is electron transfer from the excited indole to carboxylate. Electron transfer also occurs in the 80 degrees rotamer, but the major quenching process is intramolecular proton transfer. Both quenching processes are suppressed by deprotonation of the amino group. The results for cis-W3 provide compelling evidence that the complex fluorescence decay of the tryptophan zwitterion originates in ground-state heterogeneity with the different lifetimes primarily reflecting different intramolecular excited-state proton- and electron-transfer rates in various rotamers.     

Characterization of the dynamics of an essential helix in the U1A protein by time-resolved fluorescence measurements

The RNA recognition motif (RRM), one of the most common RNA-binding domains, recognizes single-stranded RNA. A C-terminal helix that undergoes conformational changes upon binding is often an important contributor to RNA recognition. The N-terminal RRM of the U1A protein contains a C-terminal helix (helix C) that interacts with the RNA-binding surface of a beta-sheet in the free protein (closed conformation), but is directed away from this beta-sheet in the complex with RNA (open conformation). The dynamics of helix C in the free protein have been proposed to contribute to binding affinity and specificity. We report here a direct investigation of the dynamics of helix C in the free U1A protein on the nanosecond time scale using time-resolved fluorescence anisotropy. The results indicate that helix C is dynamic on a 2-3 ns time scale within a 20 degrees range of motion. Steady-state fluorescence experiments and molecular dynamics simulations suggest that the dynamical motion of helix C occurs within the closed conformation. Mutation of a residue on the beta-sheet that contacts helix C in the closed conformation dramatically destabilizes the complex (Phe56Ala) and alters the steady-state fluorescence, but not the time-resolved fluorescence anisotropy, of a Trp in helix C. Mutation of Asp90 in the hinge region between helix C and the remainder of the protein to Ala or Gly subtly alters the dynamics of the U1A protein and destabilizes the complex. Together these results show that helix C maintains a dynamic closed conformation that is stable to these targeted protein modifications and does not equilibrate with the open conformation on the nanosecond time scale.     

W544F定点突变提高苏云金杆菌Cry1Ac蛋白的稳定性

W544是Cry1Ac蛋白上独特于其它Cry类蛋白的一个氨基酸, 它与F578和F604一起组成一个“螺旋桨状”的疏水簇, 通过疏水相互作用维持蛋白的三维结构稳定. 本研究通过定点突变将W544保守地替换为苯丙氨酸, SDS-PAGE分析结果表明其纯化的原毒素对紫外照射、胰蛋白酶处理和室温存贮的稳定性相对于野生Cry1Ac都有一定程度的提高; 经原子力显微镜观察, 发现W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac约长0.6 μm, 且晶体表面不及野生型光滑; 此外, W544F与野生Cry1Ac的杀虫活性相似, 但经过紫外光照射9 h后, 其保留的杀虫活性比野生型高4倍以上. W544F突变较好地解决了Cry1Ac毒素蛋白田间应用不持久的问题, 具有重要的应用价值.     

In vivo synthesized proteins with monoexponential fluorescence decay kinetics

Tryptophan, when in a protein, typically shows multiexponential fluorescence decay kinetics. Complex kinetics prevents a straightforward interpretation of time-resolved fluorescence protein data, particularly in anisotropy studies or if the effect of a dynamic quencher or a resonance energy transfer (RET) acceptor is investigated. Here, time-resolved fluorescence data are presented of an isosteric tryptophan analogue, 5-fluorotryptophan, which when biosynthetically incorporated in proteins shows monoexponential decay kinetics. Data are presented indicating that the presence of a fluoro atom at the 5-position suppresses the electron transfer rate from the excited indole moiety to the peptide bond. This process has been related to the multiexponential fluorescence decay of tryptophan in proteins. The monoexponential decay of 5-fluorotryptophan makes it possible to measure simultaneously multiple distances between 5-fluorotryptophan and a RET acceptor. We demonstrate that for an oligomeric protein, consisting of two single-tryptophan-containing subunits, the individual distances between 5-fluorotryptophan and the single substrate binding site can be resolved using a substrate harboring a RET acceptor.     

A HIGH RESOLUTION FLUORESCENCE DECAY AND DEPOLARIZATION STUDY OF HUMAN PLASMA APOLIPOPROTEINS

Abstract— Human plasma apolipoprotein A-I (apoA-I) and apolipoprotein C-I (apoC-I) were investigated by time-resolved fluorescence decay and depolarization. The tryptophyl fluorescence of apoA-I undergoes a double-exponential decay with lifetimes of 1.07 and 3.43 ns which remain unchanged over the range of apoA-I concentration studied.
The time-resolved fluorescence of both native and denatured forms of apoC-I exhibits an unusual tryptophyl fluorescence decay that was best fit to a triexponential function with lifetimes at 3.7 ± 0.2, 1.1 ± 0.1 and 0.1 ns at 2°C. The native and denatured forms of apoC-I had rotational correlation times of 1.42 and 1.19 ns at 20°C respectively. A shorter rotational correlation time associated with the internal tryptophan motions was not observed or resolved.
The decay of tryptophyl fluorescence in apoC-I/DPPC/cholesterol complex at 20°C is also triexponential with lifetimes at 4.94, 1.28 and 0.21 ns, which are longer than those of the uncomplexed forms. Two rotational correlation times of 28.32 and 0.59 ns at 20°C were resolved by fluorescence depolarization measurements. The long rotational time remained constant with temperatures above 30°C. Also, the temperature dependence of the order parameter, S², resembled a lipid phase transition curve with a transition midpoint at 38°C. The tryptophan and thus apoC-I are found to be affected by the bulk changes in the lipid.     

Nanosecond relaxation dynamics of protein GB1 identified by the time-dependent red shift in the fluorescence of tryptophan and 5-fluorotryptophan

The B1 domain of Streptococcal protein G (GB1) is a small, thermostable protein containing a single tryptophan residue. We recorded time-resolved fluorescence of the wild-type GB1 and its 5-fluorotryptophan (5FTrp) variant at more than 30 emission wavelengths between 300 and 470 nm. The time-resolved emission spectra reveal no signs of heterogeneity, but show a time-dependent red shift characteristic of microscopic dielectric relaxation. This is true for both 5FTrp and unmodified Trp in GB1. The time-dependent red shifts in the fluorescence of 5FTrp and unmodified Trp are essentially identical, confirming that the shift is caused by the relaxation of the protein matrix rather than by the fluorophore itself. The total amplitude (but not the rate) of the time-dependent red shift depends on the fluorophore, specifically, on the magnitude of the vector difference between its excited state and ground state electric dipole moments; for 5FTrp this is estimated to be about 88% of that for the unmodified Trp. The decay of the excited state fluorophore population is not monoexponential for either fluorophore; however, the deviation from the monoexponential decay law is larger in the case of unmodified Trp. The relaxation dynamics of GB1 was found to be considerably faster than that of other proteins studied previously, consistent with the small size, tightly packed core, and high thermodynamic stability of GB1.     

Quenching of tryptophan fluorescence of firefly luciferase by substrates

The interaction of firefly luciferase with substrates (luciferin and MgATP) by steady-state and time-resolved fluorescence is studied. The efficient quenching of tryptophan fluorescence of the active enzyme takes place upon its binding with substrates. In the presence of ATP the quenching is of dynamic type and is caused by structural changes in the protein molecule upon ATP binding. A model is proposed in which the complex has smaller fluorescence quantum yield than the free enzyme because of partial quenching of tryptophan fluorescence by the new microenvironment. Quenching of tryptophan fluorescence by luciferin due to the efficient energy transfer from tryptophan to luciferin is discussed. The calculated distance between Trp-419 and luciferin for the L. mingrelica luciferase in the enzyme-substrate complex is less than 12 A.     

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.     

Protein matrix elasticity determined by fluorescence anisotropy of its tryptophan residues

Rotational motions of Trp residues embedded within human hemoglobin matrix have been measured by using their steady-state fluorescence anisotropy. The mean square angular displacement theta2 of Trp residues, depending on the temperature, can be expressed by W = 1/2Ctheta2 where W is the thermal energy acting on the Trp residues and C the resilient torque constant of the protein matrix. To study the external medium influencing the protein dynamics, comparative experiments were made with protein in aqueous buffer and in the presence of 32% glycerol. The data show that between 5 degrees C and 25 degrees C, external medium acts on the protein matrix elasticity.     

FLUORESCENCE STUDIES WITH HUMAN EPIDERMAL GROWTH FACTOR

Steady-state and time-resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C-terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub-nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low-temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp-trp), it is concluded that homo-energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady-state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C-terminus segment, which is not an integral part of the three-dimensional structure of the protein. Fluorescence measurements with three site-directed mutants also show very little variation.     

Enhanced hairpin stability through loop design: the case of the protein G B1 domain hairpin

A mutational study of the peptide corresponding to the second hairpin of the protein G B1 domain (GB1p) provided a series of mutants with significantly increased fold stability. Mutations focused on improvement of the direction-reversing loop and the addition of favorable Coulombic interactions at the sequence termini. The loop optimization was based on a database search for residues that occur with the greatest probability in similar hairpin loops in proteins. This search suggested replacing the native DDATKT sequence with NPATGK, which resulted in a 4.5 kJ/mol stabilization of the hairpin fold. The introduction of positively charged lysines at the N-terminus provided an additional 2.4 kJ/mol of stabilization, affording a GB1p mutant that is 86 +/- 3% folded at 25 degrees C with a melting temperature of 60 +/- 2 degrees C. The trpzip version of this peptide, in which three of the hydrophobic core residues were mutated to tryptophan, yielded a sequence that melted at 85 degrees C. Throughout, fold populations and melting temperatures were derived from the mutation and temperature dependence of proton chemical shifts and were corroborated by circular dichroism (CD) melts. The study also suggests that the wild-type GB1p sequence is significantly less stable than reported in some other studies: only 30% folded in water at 25 degrees C.     

Intramolecular charge transfer and dielectric solvent relaxation in n-propyl cyanide. N-phenylpyrrole and 4-dimethylamino-4'-cyanostilbene

Fast intramolecular charge transfer (ICT) accompanied by dual fluorescence from a locally excited (LE) and an ICT state taking place with N-phenylpyrrole (PP) in the solvent n-propyl cyanide (PrCN) is investigated as a function of temperature between 25 and -112 degrees C. The LE and ICT fluorescence decays from -45 to -70 degrees C can be adequately fitted with two exponentials, in accordance with a two state (LE + ICT) reaction mechanism, similar to what has been observed with PP in the more polar and less viscous alkyl cyanides acetonitrile (MeCN) and ethyl cyanide (EtCN). At lower temperatures, triple-exponential fits are required for the LE and ICT decays. The ICT emission band maximum of the time-resolved fluorescence spectra of PP in PrCN at -100 degrees C displays a spectral shift from 29 230 cm-1 at t = 0 to 27 780 cm-1 at infinite time, which equilibration process is attributed to dielectric solvent relaxation. From the time dependence of this shift, in global analysis with that of the band integrals BI(LE) and BI(ICT) of the time-resolved LE and ICT fluorescence bands, the decay times 119 and 456 ps are obtained. Dielectric relaxation times of 20 and 138 ps are determined from the double-exponential spectral solvation response function C(t) of the probe molecule 4-dimethylamino-4'-cyanostilbene in PrCN at -100 degrees C. It is concluded from the similarity of the times 119 ps (PP) and 138 ps (DCS) that the deviation from double-exponential character for the fluorescence decays of PP in PrCN below -70 degrees C is due to the interference of dielectric solvent relaxation with the ICT reaction. This fact complicates the kinetic analysis of the LE and ICT fluorescence decays. The kinetic analysis for PP in PrCN is hence restricted to temperatures between -70 and -45 degrees C. From this analysis, the forward and backward ICT activation energies Ea (12 kJ/mol) and Ed (17 kJ/mol) are obtained, giving an ICT stabilization enthalpy -DeltaH of 5 kJ/mol. A comparison of the reaction barriers for PP in the three alkyl cyanides PrCN, EtCN, and MeCN (J. Phys. Chem. A 2005, 109, 1497) shows that Ea becomes smaller with increasing solvent polarity (from 12 to 6 kJ/mol), whereas Ed remains effectively constant. Both observations are indicative of a late transition state for the LE --> ICT reaction. The significance of the Leffler-Hammond postulate in this connection is discussed: not primarily the energy of the LE, ICT, and transition states but rather the extent of charge transfer in these states determines whether an early or a late transition state is present.