J. Phys. Chem. B 2008, 112, 9209–9218
9209
Effect of Side-chain Length on the Side-chain Dynamics of r-Helical Poly(L-glutamic acid) as Probed by a Fluorescence Blob Model Mark Ingratta and Jean Duhamel* Institute for Polymer Research, Department of Chemistry, UniVersity of Waterloo, 200 UniVersity AVenue West, Waterloo, ON N2L 3G1, Canada ReceiVed: March 11, 2008; ReVised Manuscript ReceiVed: April 16, 2008
Two series of pyrene-labeled poly(glutamic acid) (Py-PGA) were synthesized utilizing two different linkers for pyrene attachment, namely 1-pyrenemethylamine (PMA) and 1-pyrenebutylamine (PBA). Several PyPGAs were synthesized for each series with pyrene contents ranging from 4 to 15 mol %. Py-PGA forms a rigid R-helix in DMF that effectively locks the backbone in place, thus enabling only side-chain or linker motions to be monitored by time-resolved fluorescence. Time-resolved fluorescence decays were acquired for the pyrene monomer of the Py-PGA constructs and the fluorescence blob model (FBM) was used to quantify the dynamics of the different linkers connecting pyrene to the backbone. Nitromethane was used to shorten the lifetime of the pyrene monomer, in effect controlling the probing time of the pyrene group, from 50 to 155 ns for PGA-PBA and from 50 to 215 ns for PGA-PMA. The FBM analysis of the fluorescence decays led to the conclusion that excimer formation around the rigid R-helix backbone takes place in a compact environment. The number of glutamic acid units within a blob, Nblob, decreased only slightly with decreasing probing time for both Py-PGA constructs as a result of the compact distribution of the chromophores around the R-helix. The PGA R-helix was modeled using Hyperchem software and the ability of two pyrene groups to encounter was evaluated as they were separated by increasing numbers of amino acids along the R-helix. The number of amino acids required for two pyrenes to lose their ability to overlap and form excimer matched closely the Nblob values retrieved using the FBM. Introduction The long held view that functional proteins are polypeptides tightly packed into three-dimensional frozen objects1 has been progressively giving way to a more dynamic and plastic picture.2–4 The importance of the internal dynamics necessary for an enzyme to adopt an open or close conformation depending on whether it is in a latent or active state has been recognized.5,6 In this case, enzymatic activity is often controlled by a hinged or shear mechanism that enables large structured domains of the enzyme to generate its active site.7 More recently, this dynamic view of active proteins has been expanded into a structure continuum that ranges from the fully unstructured random coil to the fully structured protein via a partially folded molten globule.3 The extraordinary extent of protein dynamics and plasticity is illustrated during the binding of the phosphorylated kinase-inducible domain (pKID) of the transcription factor cyclic-AMP-response-element-binding-protein (CREB) onto the KID-binding domain of the CREB-binding protein (CBP). Unfolded in solution,8 pKID forms two well-defined orthogonal helices upon binding onto CBP.9 The recognition that the dynamics of selected amino acids in the protein sequence is dramatically affected as the protein switches from a dormant to an active state has led to an intense and sustained research effort aimed at characterizing polypeptide main chain and sidechain dynamics.4,6 By and large, protein main chain and side-chain dynamics have been characterized by relaxation experiments via three major techniques, namely nuclear magnetic resonance (NMR) using T1, T2, and heteronuclear NOE measurements,10–12 electron * To whom correspondence should be addressed.
paramagnetic resonance (EPR) using site-directed spin labeling (SDSL) with cysteins modified with a paramagnetic nitroxide reagent,13 or fluorescence anisotropy measurements on a tryptophan residue.14 Although different in nature, these techniques are all based on the same basic physical principle which relies on monitoring how a vector (the bond vector connecting two nuclei inducing dipole-dipole interactions for NMR, a vector along the nitroxide magnetic frame for EPR, the dipole moments for absorption and emission for fluorescence anisotropy) associated with a selected amino acid of the protein loses its original orientation over time within the reference frame of the macromolecule. The loss in orientation of the specified vector can be described via a model-free approach15–17 whereby the vector’s motion is restricted within a cone whose angle θ is related to an order parameter S (S ) 0 or 1 for freely mobile or fully hindered motion) and occurs with an effective correlation time τe. In simpler words, the magnitude and time scale of the motions experienced by an amino acid are reported by S and τe, respectively. Comparing the S and τe values obtained for the amino acids of a protein maps the dynamics experienced locally by the protein in solution.18–20 Despite being insightful and well established, experiments based on NMR, EPR, and fluorescence relaxation measurements are all subject to the same limitations. The most critical one is the impossibility of retrieving τe values much longer than the overall tumbling time (τR) of the macromolecule of interest. Residues with τe values of the same order of magnitude as τR are sometimes considered as being immobile with respect to the time scale defined by the overall tumbling of the macromolecule.18 An additional complication encountered with the fluorescence anisotropy measurements is that tryptophan is short
10.1021/jp8021248 CCC: $40.75 2008 American Chemical Society Published on Web 07/09/2008
9210 J. Phys. Chem. B, Vol. 112, No. 30, 2008 lived, which reduces the range of useful τe and τR values that can be retrieved, and often yields multiexponential decays that complicate the analysis of the fluorescence anisotropy decays.14 These complications are more likely to affect the study of sidechain dynamics, since side-chains are usually more mobile and sometimes experience slower dynamics reflected by τe values larger than 1 ns,20–22 10 ns,18 and even longer.23 Keeping these shortcomings in mind, this laboratory proposed in 2003 an alternate approach to describe the side-chain dynamics of structured polypeptides that would be independent of τR.24 This approach departed from the relaxation principles traditionally used in NMR, EPR, or fluorescence anisotropy. It was based on fluorescence dynamic quenching measurements which were, as such, insensitive to the magnitude of τR. These experiments were conducted by randomly labeling a polypeptide of interest with a chromophore and its quencher and monitoring the chromophore-quencher (C-Q) encounters with a Fluorescence Blob Model (FBM).25 Information about side-chain dynamics was retrieved from the size of a blob, Nblob, where C-Q encounters occur and the rate constant describing the encounters between one chromophore and one quencher inside the same blob, kblob. In other words, a comparison can be drawn between the parameters S and Nblob, which are a measure of the volume probed by the side-chain given a fixed amount of time, and τe and kblob, which are a measure of the rate at which the side-chain moves within that volume. Additionally, the Nblob parameter provides the added benefit of altering in a controlled manner the volume probed by the side-chain by adjusting the lifetime of the chromophore.26 The first FBM experiments conducted on structured polypeptides were carried out by randomly labeling poly(L-glutamic acid) (PGA) with 1-pyrenemethylamine (PMA) to yield PGAPMA and monitoring the process of excimer formation between pyrene pendants.24 Excimer formation is an example of dynamic fluorescence quenching where quenching of an excited pyrene occurs via diffusive encounter with a ground-state pyrene to form an excimer.27 To ensure solubilization of pyrene which is insoluble in aqueous solution, these experiments were conducted in N,N-dimethylformamide in which PGA has been shown to adopt an R-helical conformation.28 An excellent agreement was found between the Nblob value retrieved with the FBM for the PGA-PMA constructs and the distance spanned by a pyrenyl pendant predicted by molecular mechanics optimization. Despite this early success and in order to demonstrate the viability of the FBM approach as a robust alternative to the well-established model-free approach,15–17 the FBM must be shown to be sensitive enough to probe changes in polypeptide side-chain dynamics induced by subtle modifications made at the molecular level to the side-chains. This issue was addressed herein by using the FBM to compare the process of excimer formation in PGAPMA and PGA-PBA, where the PGA-PBA constructs were prepared by randomly labeling PGA with 1-pyrenebutylamine (PBA). The structure of the PGA-PMA and PGA-PBA constructs enables the comparison of the FBM response when the linker connecting pyrene to the PGA R-helix is lengthened by three methylene units (Scheme 1). The experiments reported in this study demonstrate that the FBM analysis of fluorescence dynamic quenching experiments is sensitive enough to accurately probe at the molecular level subtle changes induced by a monomethylene and tetra-methylene linker used to connect pyrene to the PGA backbone. These experiments also provide quantitative information on the volume probed by an excited chromophore attached to a structured polypeptide. To the best of our knowledge, they represent the
Ingratta and Duhamel SCHEME 1: Structure of PGA-PMA and PGA-PBA; n is Equal to 1 and 4, Respectively
first attempt in the literature at probing the sensitivity of a technique that does not rely on relaxation experiments to characterize the dynamics of the side-chains of a biological macromolecule. Experimental Section Materials. Chemicals were purchased from Sigma-Aldrich (Milwaukee, WI) and used as received unless otherwise stated. Distilled in glass N,N-dimethylformamide (DMF) was purchased from Caledon Laboratories (Georgetown, ON) and used as received. Two batches of PGA-sodium salt were purchased from Sigma-Aldrich with the following information: Batch 1: DP (viscosity) ) 333, MW (viscosity) ) 50.3 kg/mol, DP (MALLS) 141, MW (MALLS) 21.3 kg/mol. Batch 2: DP (viscosity) ) 648, MW (viscosity) ) 97.8 kg/mol, DP (MALLS) 274, MW (MALLS) 41.4 kg/mol. This range in molecular weights is not expected to affect the FBM parameters since the fluorescence experiments characterize the behavior of a pyrene inside a blob whose dimensions are usually much smaller than those of the polymer.29 The largest blob found in this study was made of 31 glutamic acids equivalent to a blob molecular weight of 4 000 g · mol-1, indeed substantially smaller than the PGA samples. 4-(1-Pyrene)butylamine (PBA) was purchased from Toronto Research Chemicals (Toronto, ON). Synthesis of PGA-PMA and PGA-PBA. The synthesis and purification of pyrene-labeled PGA has been described elsewhere.24 The only change made to the labeling procedure from ref 24 was the addition of two extra dialysis washes for the PGA-PBA samples to remove free PBA. The PGA-PBA solutions were first dialyzed twice against a 1:1 DMF/water mixture for 3 h, followed by 4 days of dialysis against aqueous solutions following the same protocol as in ref 24. The structures of PGA-PMA and PGA-PBA are shown in Scheme 1. Pyrene Content Determination. The pyrene content, λPy, expressed in micromoles of pyrene per gram of polymer in the salt form (µmol/g), was determined using eq 1. The sodium salt of the pyrene-labeled PGA (PyPGNa) was dried using a Labconco Freezone 6 freeze drier prior to careful weighing of the polymer.30 A mass, m, of freeze-dried PyPGNa was weighed and then dissolved in water where it was acidified using 1N HCl. The aqueous solution was then evaporated under a gentle flow of N2 before the dry Py-PGA was subsequently dissolved in a known volume of DMF, V. The pyrene concentration, [Py], was determined by UV-vis absorption measurements using Beer-Lambert’s Law applied to the pyrene absorption at 346 nm with an extinction coefficients of 40 000 M-1 · cm-1 for PGA-PMA and 36 000 M-1 · cm-1 for PGA-PBA, determined from the model compounds 1-pyrenemethanol and 1-pyrenebutanol, respectively. The absorption measurements were conducted in DMF, an organic solvent that prevents the distortion of the absorption spectra observed in aqueous solutions due to the formation of pyrene aggregates.31
Side-chain Dynamics of R-Helical Poly(L-glutamic acid)
λPy )
[Py] m⁄V
J. Phys. Chem. B, Vol. 112, No. 30, 2008 9211
(1)
Steady-state Fluorescence Measurements. All fluorescence spectra were acquired on a PTI fluorometer using the usual right angle geometry with a 346 nm excitation wavelength. All PGAPMA and PGA-PBA solutions were prepared in DMF with polymer concentrations below 2 × 10-6 M to avoid intermolecular excimer formation. Solutions were degassed for 20 min by bubbling a gentle flow of N2 to remove oxygen. The monomer (IM) and excimer (IE) intensities were obtained by integrating the fluorescence spectra between 373-379 nm and 500-530 nm for the monomer and excimer, respectively. Time-Resolved Fluorescence Measurements. Monomer fluorescence decays were acquired for PGA-PMA and PGAPBA solutions in DMF with various nitromethane concentrations (Table 1) using an IBH 5000F coaxial nanosecond flash lamp filled with H2 gas with an excitation at 346 nm and emission at 376 nm. Excimer fluorescence decays were also acquired for PGA-PMA and PGA-PBA solutions in DMF with no quencher by exciting at 346 nm and collecting the emission at 510 nm. All decays were collected over 1024 channels with up to 20 000 counts at the peak maximum for the lamp and decay curves. The instrument response function was determined by applying the MIMIC method32 to the lamp reference decays obtained with PPO [2,5-diphenyloxazole] in cyclohexanol (τ ) 1.42 ns) and BBOT [2,5-bis(tert-butyl-2-benzoxazolyl)thiopene] in ethanol (τ )1.47 ns) for the monomer and excimer decays, respectively. The solutions were prepared in the same way as for steadystate fluorescence measurements. Analysis of the Fluorescence Decays. The monomer and excimer fluorescence decays were fit with a sum of exponentials as shown in eq 2. The last exponential of the sum handles the unquenched pyrene monomers that emit with a lifetime τM whereas the other exponentials represent those pyrene monomers that form excimer by diffusion. Fitting the monomer decays with eq 2 yields a measure of the molar fraction of unquenched monomers, fMfree ) aM/(Σai + aM). The molar fraction of pyrene monomers forming excimer by diffusion is given by fMdiff ) 1 - fMfree. The monomer decays were also fit using eq 3, which is based on the FBM analysis of the excimer kinetics.25,29 Within the framework of the FBM, the polymer coil is compartmentalized into blobs among which the pyrenes randomly attached to the polymer distribute themselves according to a Poisson distribution. The kinetics of excimer formation between pyrenes is handled in the same manner as if the blobs were surfactant micelles. Consequently, eq 3 bears a strong resemblance with the equations that have been derived to describe the timedependent concentration profile of chromophores located in surfactant micelles loaded with quenchers.33,34 Over the past ten years, the FBM has been used to study the dynamics of several polymers in dilute solution.24,25,29,35–37 nexp
i(t) )
∑ ai exp(-t ⁄ τi) + aM exp(-t ⁄ τM) i)1
[(
* ](t)0) exp - A2 + [Py*](t) ) [Pydiff
]
)
with nexp ) 2, 3 (2)
1 t - A3 × τM
(1 - exp(-A4t)) + [Py*free](t)0) exp(-t/τM) (3) The parameters A2, A3, and A4 used in eq 3 are expressed in eq 4 as a function of the FBM parameters kblob, , and ke[blob].
A2 ) 〈n〉
kblobke[blob] kblob + ke[blob] A3 ) 〈n〉
k2blob (kblob + ke[blob])2 A4 ) kblob + ke[blob] (4)
The first exponential in eq 3 describes the behavior of the pyrene monomers that form excimer by the diffusive encounter of an excited pyrene and a ground-state pyrene. The unquenched lifetime of the pyrene monomer, τM, is determined from the monomer fluorescence decay of a polymer sparingly labeled with pyrene (pyrene content 100 ns), kdiff is expected to remain constant with probing time, as reflected by the similar values taken by koblob for PGA-PMA and PGA-PBA in Figure 4. Since koblob is expected to scale as (Noblob)3 according to the PGA-PBA trend in Figure 7, and since Noblob is 40% larger for PGA-PBA than for PGA-PMA, kdiff for PGA-PBA is expected to be 2.7 larger than for PGA-PMA as expected from the more flexible nature of the PBA linker.48 Meaning of the Nblob Parameter. A considerable benefit of the well-defined R-helical structure of pyrene-labeled PGA constructs is the ability to compare the experimental Noblob obtained using the FBM analysis to the known physical dimensions of a PGA R-helix. To this end, Hyperchem software (version 7.04) was used to create a 40 unit PGA R-helix labeled with two PMA or two PBA following a protocol which has been described in an earlier publication.24 One pyrene group
9216 J. Phys. Chem. B, Vol. 112, No. 30, 2008
Ingratta and Duhamel
Figure 8. An illustration of the ability of two pyrene groups to overlap when separated by 17 Glu. Top: PGA-PBA; good overlap. Bottom: PGA-PMA; no overlap.
Figure 9. Pyrene carbon-overlap as a function of the number of glutamic acid units between pyrene groups. PGA-PBA (2), PGA-PMA (4).
was first attached at the eighth glutamic acid, while the second pyrene was attached at the ninth glutamic acid. Molecular mechanics optimizations were performed on this construct with the Fletcher-Reeves algorithm in order to bring the plane of the two pyrenes within 3.4 Å from each other (Figure 8).24 During optimization, only the pyrene groups and PGA sidechains were allowed to move while the backbone was held rigid. Keeping one pyrene attached on the eighth glutamic acid, the second was then attached on a second glutamic acid located at position no. 9, 10, 11..., and the optimization was conducted for each Py-PGA construct. The extent of pyrene-pyrene overlap was completed by counting the number of carbon atoms from the first pyrene that would be covered by the area of the second pyrene. The number of overlapping carbons was determined for PGA-PMA and PGA-PBA and is shown as a function of the number of glutamic acids separating the two pyrenes in Figure 9. The trends shown in Figure 9 indicate that the overlap between two pyrenes worsens when the two pyrenes are separated by more than 11 and 17 Glu residues for PGAPMA and PGA-PBA, respectively. Also, the longer reach of the butyl linker of PGA-PBA enables a good overlap between the pyrene moieties over a longer stretch of R-helical PGA. In
these simulations, a good overlap between two pyrenes is characterized by one pyrene having at least 7 C-atoms covered by the area of the second pyrene. The number of overlapping carbons was consistently larger for PGA-PBA than for PGA-PMA and this with any number of Glu residues separating the two pyrene groups, even for those pyrenes that were only separated by a few amino acids (Figure 9). The increased capacity of the pyrene groups of PGA-PBA to overlap is due to the longer, more flexible butyl spacer that enables more rearrangements around the PGA R-helix than the shorter methylene linker of PGA-PMA can afford. With a probing time of 155 ns which represents the longest probing time where Noblob could be determined for both Py-PGA constructs (Table 1) and noting that Noblob does not vary much with probing time (Figure 6), Noblob values of 22 and 31 glutamic acids were obtained experimentally using the FBM for PGAPMA and PGA-PBA, respectively. Assuming that a PGA blob has an excited pyrene at its center, it would reach a groundstate pyrene located Noblob/2 amino acids upward or downward the R-helix.24 According to this statement, the maximum distance separating two pyrenes where they fail to overlap equals Noblob/2 ) 11 for PGA-PMA or 15 for PGA-PBA. These Noblob/2 values obtained experimentally from a FBM analysis of the fluorescence decays stand in very good agreement with the values of 11 and 17 predicted for, respectively, PGA-PMA and PGA-PBA from the data shown in Figure 9 and obtained via molecular mechanics optimizations. Comparing Relaxation Techniques and Fluorescence Dynamic Quenching. Information on side-chain dynamics is typically obtained from NMR, EPR, or fluorescence anisotropy measurements.11–14 As argued in the Introduction, all these techniques are based on the same concept, namely that the residue of interest moves unhindered with a correlation time τe and within a cone whose angle is defined by an order parameter S. S and τe give information about the magnitude and time scale of the motions undergone locally by the residue. Despite being conceptually different, the FBM retrieves somewhat similar information where the magnitude and time scale of the local motion are inferred from Nblob and kblob. The magnitude and time scale for local motions are now compared whether they are obtained by relaxation or fluorescence quenching experiments.
Side-chain Dynamics of R-Helical Poly(L-glutamic acid) A measure of the time scales describing the pyrene-pyrene encounters is obtained by taking koblob-1 which ranges from 17 to 45 ns according to the values listed in Figure 4. At first glance, such large time scales are difficult to reconcile with the subnanosecond τe values typically retrieved from NMR relaxation studies conducted on the N-H bond of the polypeptide backbone,19–22 although a few examples report τe values obtained by NMR for side-chain motion that are greater than 1 ns.20–22 This discrepancy is certainly a result of the different positions of the probe on the macromolecule. An N-H bond locked in the structured polypeptide backbone can only experience rapid motions which are reported by the NMR relaxation experiments. By comparison, the probe of the Py-PGA constructs is held far away from the polypeptide backbone, so that a much larger volume is being scanned by the probe resulting in much slower motions probed by fluorescence. Supporting this notion is the observation that τe values in the 1-20 ns range have been reported by EPR relaxation experiments conducted on a paramagnetic nitroxide moiety held 4 atoms away from the polypeptide backbone.18 These observations suggest that a longer linker between a probe and the polypeptide backbone enables the probe to scan a larger volume which results in longer time scales, as observed for EPR relaxation18 and now fluorescence dynamic quenching experiments. Limitations of the FBM. This study demonstrates that sidechain dynamics of structured polypeptides can be probed efficiently by the FBM, a model that does not rely on and is not subject to the same limitations of the typical relaxation processes used in NMR, EPR, or fluorescence anisotropy measurements. Although these results are encouraging, the procedure in its present state has several flaws that will need to be overcome if it is to obtain the same appeal as the modelfree approach. First of all, FBM studies rely heavily on pyrene, a hydrophobic chromophore which aggregates in aqueous solution. To apply this method to study side-chain dynamics of structured polypeptides in water, water-soluble chromophore/ quencher pairs must be found. Second, whereas random labeling can be easily done with PGA, it will be much more difficult to achieve with a specific peptide sequence. Since altering an amino acid sequence with a fluorophore might alter the structure of interest, clever labeling schemes will need to be designed. Unless these hurdles are circumvented, studies of side-chain dynamics based on the FBM will be limited to model systems such as PGA. Conclusions Two Py-PGA constructs were prepared where the linker connecting pyrene to the polypeptide backbone was made of 5 or 8 atoms for PGA-PMA or PGA-PBA, respectively. The dynamics and amplitude of the motions of the pyrene pendants were characterized by studying their ability to form an excimer. To this end, the monomer fluorescence decays of the Py-PGA solutions were acquired and analyzed with the FBM. Information about the side-chain dynamics and amplitude was obtained with koblob and Noblob, respectively. Interestingly, the rather minor change in the linker length from 5 to 8 atoms was clearly probed by the FBM, resulting in a concomitant increase of Noblob from 22 to 31 glutamic acids. The size of a PGA blob found by fluorescence was also determined from the cutoff distance estimated by molecular mechanics optimizations over which encounters between two pyrene moieties would be prevented by the spacing separating the two glutamic acids bearing the pyrenes. These optimizations resulted in Noblob values of 11 × 2 + 1 ) 23 and 17 × 2 + 1 ) 35, in excellent agreement with those of 22 and 31 obtained experimentally with the FBM.
J. Phys. Chem. B, Vol. 112, No. 30, 2008 9217 The dynamics of the pyrenyl side-chains were described by the rate constant kblob whose expression is given in eq 7 as the ratio kdiff/Vblob. Comparison of the values of ln(koblob) and ln(Noblob) in Figure 7 led to two important conclusions. First, a scaling relationship koblob ∝ (Noblob)R was found where R equaled 2.9 ( 0.5 and 4.6 ( 1.3 for PGA-PBA and PGA-PMA, respectively. These R-values are much larger than those of 1.5 or 1.8 found for random polymer coils26 and they reflect the more compact nature of the PGA R-helix. Second, the R-value of 4.6 found for PGA-PMA was larger than the maximum possible value of 3.0, which implies that kdiff increases as the pyrene lifetime decreases. This observation indicates that the shorter and more rigid linker of PGA-PMA induces some strain on the motion of the pyrenes. With longer lifetimes (τM > 100 ns), the linker is allowed enough time to probe those more strained conformations resulting in a smaller koblob value. As the lifetime is shortened, the pyrenes can only probe those conformations which are less strained and koblob takes a larger value. This study has demonstrated the outstanding sensitivity of the fluorescence dynamic quenching experiments based on pyrene excimer formation. Lengthening the side-chains connecting pyrene to the PGA R-helix by three methylene units is probed efficiently by these fluorescence measurements. The procedure used in this report to describe the diffusional encounters between pyrene pendants along the helix provides an alternate method to study side-chain dynamics of structured polypeptides which is not based on the relaxation experiments typically used in NMR, EPR, and fluorescence anisotropy. Acknowledgment. M.I. and J.D. are indebted to funding from the Natural Science and Engineering Research Council of Canada, an Ontario’s Premier Research Excellence Award, and the Canadian Foundation for Innovation. Supporting Information Available: Figures SI.1 and SI.2, plots of molar ellipticity versus wavelength; Figure SI.3, plot of molar ellipticity versus pyrene content; Figure SI.4, plot of k1[M] versus concentration of two pyrene derivatives; Figure SI.5, plot of τo/τ versus nitromethane concentration; Figure SI.6, examples of fits of the pyrene-labeled PGAs; Tables SI.1-4, decay times and pre-exponential factors obtained from the analysis of the fluorescence decays with a sum of exponentials; Tables SI.5-6, parameters retrieved from the FBM analysis of the Py-PGA fluorescence decays. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Pauling, L. Nature 1948, 161, 707–709. (2) Tompa, P. FEBS Lett. 2005, 579, 3346–3354. (3) Dyson, J.; Wright, P. E. Nature ReV. Mol. Cell Biol. 2005, 6, 197– 208. (4) Lindorff-Larsen, K.; Best, R. B.; DePristo, M. A.; Dobson, C. M.; Vendruscolo, M. Nature 2005, 433, 128–132. (5) Hammes, G. G. Biochemistry 2002, 41, 8221–8228. (6) Benkovic, S. J.; Hammes-Schiffer, S. Science 2003, 301, 1196– 1202. (7) Gerstein, M.; Lesk, A. M.; Chothia, C. Biochemistry 1994, 33, 6739–6749. (8) Richards, J. P.; Ba¨chinger, H. P.; Goodman, R. H.; Brennan, R. G. J. Biol. Chem. 1996, 271, 13716–13723. (9) Radhakrishnan, I.; Pe´rez-Alvarado, G. C.; Parker, D.; Dyson, H. J.; Montminy, M. R.; Wright, P. E. Cell 1997, 91, 741–752. (10) Ishima, R.; Torchia, D. A. Nat. Struct. Biol. 2000, 7, 740–743. (11) Dyson, H. J.; Wright, P. E. Chem. ReV. 2004, 104, 3607–3622. (12) Igumenova, T. I.; Frederick, K. K.; Wand, A. J. Chem. ReV. 2006, 106, 1672–1699. (13) Fanucci, G. E.; Cafiso, D. S. Curr. Opin. Struct. Biol. 2006, 16, 644–653. (14) Bucci, E.; Steiner, R. F. Biophys. Chem. 1988, 30, 199–224.
9218 J. Phys. Chem. B, Vol. 112, No. 30, 2008 (15) Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546–4559. (16) Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4559–4570. (17) Szabo, A. J. Chem. Phys. 1984, 81, 150–167. (18) Lietzow, M. A.; Hubbell, W. L. Biochemistry 2004, 43, 3137–3151. (19) Spyracopoulos, L.; Lewis, M. J.; Saltibus, L. F. Biochemistry 2005, 44, 8770–8781. (20) Kemple, M. D.; Buckley, P.; Yuan, P.; Prendergast, F. G. Biochemistry 1997, 36, 1678–1688. (21) Skrynnikov, N. R.; Millet, O.; Kay, L. E. J. Am. Chem. Soc. 2002, 124, 6449–6460. (22) Millet, O.; Mittermaier, A.; Baker, D.; Kay, L. E. J. Mol. Biol. 2003, 329, 551–563. (23) Tcherkasskaya, O.; Ptitsyn, O. B.; Knutson, J. R. Biochemistry 2000, 39, 1879–1889. (24) Duhamel, J.; Kanagalingam, S.; O’Brien, T.; Ingratta, M. J. Am. Chem. Soc. 2003, 125, 12810–12822. (25) Duhamel, J. Acc. Chem. Res. 2006, 39, 953–960. (26) Kanagalingam, S.; Spartalis, J.; Cao, T.-M.; Duhamel, J. Macromolecules 2002, 35, 8571–8577. (27) Birks, J. B. Photophysics of Aromatic Molecules; Wiley: New York, 1970. (28) Yamaoka, K.; Ueda, K. J. Phys. Chem. 1982, 86, 406–413. (29) Mathew, H.; Siu, H.; Duhamel, J. Macromolecules 1999, 32, 7100– 7108. (30) In previous work by Duhamel et al.,24 the Py-PGA was purified using dialysis against aqueous solution. The aqueous solution was removed by rotary evaporation, and the polymer film was further dried under vacuum at 40 °C prior to pyrene content determination. In the current work, a Labconco freeze-drying system was used to recover the purified Py-PGA from aqueous solution. This difference has proven essential for accurate pyrene content determination, as it was determined that simple vacuum drying did not remove all of the water from the polymer sample. Pyrene contents determined for the previous “wet” polymer samples were incorrect in some cases given that the mass of the polymer was actually lower than the amount weighed. This resulted in under-estimated pyrene content for some Py-PGAs. The pyrene content has the largest effect on the Nblob o parameters reported in ref 24 as calculated by eq 6. The Nblob value for Py-PGA reported in ref 24 dropped from 32 ( 1 to 27 ( 1 after the pyrene content of the Py-PGA samples had been re-determined after freeze-drying o the samples. The Nblob value of 27 ( 1 obtained with our 25 year old PRA
Ingratta and Duhamel system24 is similar to that of 24 ( 1 obtained in the current work by analyzing the fluorescence decays acquired with our recently purchased IBH time-resolved fluorometer. (31) Winnik, F. M. Chem. ReV. 1993, 93, 587–614. (32) James, D. R.; Demmer, D. R.; Verall, R. E.; Steer, R. P. ReV. Sci. Instrum. 1983, 54, 1121–1130. (33) Tachiya, M. Chem. Phys. Lett. 1975, 33, 289–292. (34) Infelta, P. P.; Graetzel, M.; Thomas, J. K. J. Phys. Chem. 1974, 78, 190–195. (35) Ingratta, M.; Duhamel, J. Macromolecules 2007, 40, 6647–6657. (36) Kanagalingam, S.; Ngan, C. F.; Duhamel, J. Macromolecules 2002, 35, 8560–8570. (37) Irondi, K.; Zhang, M.; Duhamel, J. J. Phys. Chem. B 2006, 110, 2628–2637. (38) Press, W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. Numerical Recipes. The Art of Scientific Computing (Fortran Version); Cambridge University Press: Cambridge, 1992. (39) Martinho, J. M. G.; Egan, L. S.; Winnik, M. A. Anal. Chem. 1987, 59, 861–864. (40) Shoji, O.; Ohkawa, M.; Kuwata, H.; Sumida, T.; Kato, R.; Annaka, M.; Yoshikuni, M.; Nakahira, T. Macromolecules 2001, 34, 4270–4276. (41) Volumes were calculated using Quantitative Structure Activity Relationship (QSAR) Properties Software v7.0, Hypercube Inc., as part of the Hyperchem 7.0 package. For the calculation, the pyrene was bound by its Van der Waals surface. (42) Lakowicz, Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (43) Winnik, M. A.; Egan, L. S.; Tencer, M.; Croucher, M. D. Polymer 1987, 28, 1553–1560. (44) Cuniberti, C.; Perico, A. Eur. Polym. J. 1980, 16, 887–893. (45) Ingratta, M.; Duhamel, J. submitted for publication. (46) de Gennes, P.-G. Scaling Concepts in Polymer Physics; Cornell University Press: Ithaca, NY, 1979. (47) Ingratta, M. Aspects of Polymer Chain Dynamics in Solution Studied by Fluorescence. Ph.D. Thesis, University of Waterloo, Waterloo, Ontario, 2008. (48) The value of 2.7 is found from (1.4)3 ) 2.7 where 1.4 is the ratio o o Nblob PGA-PBA)/Nblob (PGA-PMA).
JP8021248
