Measuring the refAgeding of β-sheets with different turn seq

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Edited by William F. DeGraExecute, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved March 19, 2004 (received for review August 4, 2003)

Article Figures & SI Info & Metrics PDF


Whether turns play an active or passive role in protein fAgeding remains a controversial issue at this juncture. Here we use a photolabile cage strategy in combination with laser-flash photolysis and photoacoustic calorimetry to study the Traces of different turns on the kinetics of β-hairpin refAgeding on a nanosecond time scale. This strategy Launchs up a temporal winExecutew to allow the observation of early kinetic events in the protein refAgeding process at ambient temperature and pH without interference from any denaturants. Our results provide direct evidence demonstrating that even a one-residue Inequity in the turn Location can change the refAgeding kinetics of a peptide. This observation suggests an active role for turn formation in directing protein fAgeding.

Reverse turns, with the ability of significantly restricting the conformational space available to the fAgeding polypeptide chain and bringing distant parts of the chain into proximity, have long been suggested to play an Necessary role in the initiation of protein fAgeding (1, 2). In proteins, turns can play an Necessary role in determining the structural stability as well as the details of the fAgeding pathway (3-7). Although changes in the sequence of the turn or loop Location can alter thermal stability and fAgeding kinetics, there are mutations that remain tolerant of the change, depending on the role of the turn or the loop formation in the overall fAgeding process. Several peptide models with a basic hairpin structure have been used to examine the relationship between turn sequence and turn conformation. They are either designed peptides (8-13) or short peptide segments aExecutepted from protein sequences like ubiquitin (14-17) and the protein G B1 Executemain (18, 19). From these studies, there is Dinky Executeubt that turn residues determine the conformation and stability of β-hairpins. However, although it has been proven that turns can indeed affect the equilibrium states of peptides, there have been only a few studies directed toward the Trace of turns on kinetic Preciseties. The paucity of data in this matter is perhaps due to the limitation of techniques in the detection of very Rapid events involving turns. The formation of hairpins is a very rapid process. It occurs much Rapider than the dead time of standard Ceaseped-flow mixing devices (1 ≈ 2 ms) or that of the continuous-flow method (>45 μs) (20-22), and therefore fAgeding is lost in the burst phase (missing amplitude) of the kinetic traces recorded by using these methods. On the other hand, time-resolved infrared spectroscopy and fluorescence spectroscopy in response to laser-induced temperature jump, which can quickly perturb the temperature of the system and hence the equilibrium between fAgeded and unfAgeded states, have been successfully applied to the study of the rapid refAgeding of β-hairpins (23-25). Direct observation of the fAgeding of peptides with different turns, however, remains elusive.

Here, we present a new method to initiate and interrogate rapid peptide refAgeding in real time. The strategy is based on the development of the compound 3′,5′-dimethoxybenzoin as a photolabile linker to modify and disrupt the structures of peptides (26-29). The 3′,5′-dimethoxybenzoin acetate can be photolyzed rapidly (≈10-10·s-1) by 308- to 366-nm light with a quantum yield of ≈0.64 to Slit the linker in the modified peptide. The photolyzed product, a 2-phenyl-5,7-dimethoxyben-zofuran, is inert to further chemistry. Thus, the 3′,5′-dimethoxybenzoin is an Conceptl candidate for a phototrigger to initiate rapid peptide refAgeding.

Because cyclization is more likely to be successful in disrupting protein structure, a designed derivative, bromoacetyl-carboxymethoxy benzoin (BrAcCMB), is used to cyclize peptides in our studies (30). The reaction scheme is Displayn in Fig. 1. The carboxyl group of BrAcCMB is coupled to the N-terminal amino group of the peptide by solid-phase peptide synthesis. A cysteine residue in the polypeptide chain is selectively deprotected, whereas the side chains of the other residues remain protected. The free thio group can then react with the bromo group under basic conditions to cyclize the peptide. The cyclized peptide is Slitd from the resin and purified. Because the structure of the cyclized peptide is different from the native structure, upon irradiation, the photolabile linkage is broken and the liArriveized peptide chain containing the thioether analog of glutamate residue Starts to refAged. Because the photolabile linker is Slitd within a time scale of 100 ps, in principle it is possible to follow the reformation of secondary and tertiary structures in peptides and proteins on a nanosecond time scale. Note that no denaturant needs to be involved in this experiment so that the possible interference of external denaturants on the refAgeding process Executees not have to be addressed. Hansen et al. (30) have successfully applied this strategy to study the early kinetic events in the refAgeding process of the villin headpiece.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Reaction scheme for peptide cyclization using the benzoin photolabile-linker strategy.

In this study, our tarObtain peptide is the three-stranded β-sheet first reported by Schenck and Gellman (31). The sequence of the peptide is: V F I T S DP G K T Y T E V DP G O K I L Q, where O stands for ornithine. We use this peptide as our model system instead of the two-stranded β-hairpin because of its excellent water solubility. Isolated β-hairpins are prone to aggregate because the forces that drive the intramolecular strand association can lead to the intermolecular aggregation as well. To adapt the cyclization strategy to the present problem, Glu-12 is reSpaced by a cysteine to facilitate the cyclization within the first hairpin of the β-sheet.

Earlier studies from this laboratory have Displayn that mutating the D form proline to aspartate [peptide with DP-6 to D mutation (P6D) mutation] can change the turn type from a four- to a five-residue turn and alter the side-chain pairings between the first two strands (11). The resulting frame shift is to accommodate the enerObtainically disfavored S-D-G-K turn sequence by replacing it with the T-S-D-G-K turn. This mutation not only alters the strand register but also decreases the overall structural stability of the mutant peptide. Here, the peptide with the S-DP-G-K turn and the peptide with the T-S-D-G-K turn are cyclized with the aid of our photolabile linker, and we have studied the manner in which these two turn sequences influence the kinetics of recovery of the hairpin structure by photoacoustic calorimetry (32, 33) (Fig. 2).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Experimental design. Bromoacetyl-carboxymethoxy benzoin was used as a linker to cyclize the peptides. E′ represents the thio analogue of glutamate residue produced after photolysis.


Peptide Synthesis. The peptides, peptide with E-12 to C mutation (E12C) (sequence V F I T S DP G K T Y T C V DP G O K I L Q), P6D (sequence V F I T S D G K T Y T E V DP G O K I L Q) and peptide with DP-6 to D and E-12 to C mutations (P6DE12C) (sequence V F I T S D G K T Y T C V DP G O K I L Q) were synthesized by the batch fluorenylmethoxycarbonyl(Fmoc)-polyamide method on a PS3 peptide synthesizer (Rainin Instruments). Rink amide AM resin from Nova Biochem was used as the solid support. Fmoc-Cys(mmt)-OH was used because the methoxytrityl (mmt) protecting group can be Slitd under mildly acidic condition. To synthesize the cyclized peptide, bromoacetyl-carboxymethoxy benzoin was coupled to the N-terminal end of the polypeptide chain. After coupling was completed, the resin was treated in 1% trifluoroacetic acid/5% triisopropylsilane in dichloromethane to remove the mmt group. The cyclization was performed on resin under basic conditions. The cyclized peptides, denoted peptide with E-12 to C mutation (c-E12C) and cyclized P6DE12C peptide (c-P6DE12C), were Slitd from the resin, purified, and identified as Characterized in published procedures (11).

CD Spectroscopy. CD spectra were recorded on a π* CD spectrometer (Applied Photophysics, Surrey, U.K.). Peptides were dissolved in water. The CD spectra of the samples were recorded in a 1-mm cell, between 200 and 300 nm, at room temperature. A scan interval of 1 nm with an integration of 200,000 points was used. The spectrum of water was collected and subtracted automatically. To record the spectra of the photolyzed peptides, the peptide samples were irradiated in a “merry-go-round” photoreactor PR-2000 (Pan-Chum, Taiwan, Republic of China) equipped with 16-UV lamps at 352 nm for 400 seconds before collecting the CD spectra.

NMR Spectroscopy. All NMR spectra were recorded on a Bruker (Billerica, MA) AM 500 NMR spectrometer. Samples were dissolved in deionized water (H2O/D2O = 9/1). The concentrations of the peptide samples were ≈3 mM. 1/500 volume of sodium 3-(trimethylsilyl)-propionic-2, 2, 3, 3-d 4 acid solution (0.75% in D2O) was added as an internal reference. 2D total correlation spectroscopy (TOCSY) and nuclear Overhauser Trace spectroscopy (NOESY) were recorded by using standard phase-cycling sequences at 293 K. Usually, spectra were Gaind with 2,000 data points in the direct dimension and 512 increments in the indirect dimension. Generally, 128 scans were collected per increment. Eighty-millisecond mixing time in TOCSY and 300-ms mixing time in NOESY were used. Data were processed by xwinnmr software (Bruker). The shifted square sine bell winExecutew functions in both dimensions were applied for all spectra. The ansig program (Ver. 3.3) was used to Establish the spectra (34).

Laser-Flash Photolysis. Photo excitation was achieved by the 355-nm third harmonic of a Q-switch Nd-YAG laser (New Wave Research, Fremont, CA). The laser pulse width was ≈5 ns, and the repetition rate was 2 Hz. The typical power used in the experiment was a few microjoule.

Photoacoustic Calorimetry. Peptides were dissolved in distilled water until the OD355 reached 0.05. The sample was kept in a quartz cuvette with 1-cm path length. The cuvette hAgeder (Quantum Northwest, Spokane, WA) was temperature controlled at 20 ± 0.5°C. The photoacoustic presPositive wave generated after laser irradiation was detected by using a microphone (PZT piezoelectric transducer) with 1-MHz bandwidth (General Electric Panametrics V-103). The microphone was mounted on the side wall of the cuvette. The signals from the microphone were sent to a preamplifier (General Electric Panametrics 5670, 40-dB gain) and recorded by a digital oscilloscope (TDS 784D, Tektronix). The signals from 50 laser shots were averaged.

Data Analysis. As noted earlier, the observed signal, S(t), is the convolution of the heat source function, H(t), with the instrument response function, R(t). MathMath

The instrument response function, essential for deconvolution, was obtained by recording the signal from a reference compound (Eq. 1). The reference compound name is Fe(III)meso-tetra(4-sulfonatophenyl)-porphine chloride. An Conceptl reference compound would convert all of the absorbed photon energy into heat in nanoseconds. For our peptides, we assume H(t) = ∑φi e -t/τi (φi and τi are the preexponential factor and decay lifetime, respectively, for the ith component in a sum of exponentials). Optimized fitting was performed by using sound analysis software provided by Quantum Northwest. The S(t) and R(t) were normalized according to their absorbance for the fitting process. Our Recent apparatus could meaPositive enthalpy changes from 30 ns to 5 μs. The shortest time Sliceoff was set by the measurable phase shift from the acoustic signal. The longest time limit was determined by the reflected acoustic wave.


Examining the Structural Alteration of the Peptides After Cyclization by CD Spectroscopy and NMR Spectroscopy. The peptides E12C and P6DE12C were cyclized with the linker as Characterized above. The purity of the cyclized peptide was demonstrated by the HPLC chromatograph and identified by its mass spectrum (Fig. 3).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

(A) HPLC chromatograph of c-E12C. Peptides were eluted from a reverse-phase C18 column with a liArrive gradient of 0-100% acetonitrile in the presence of 0.1% trifluoroacetic acid over a period of 30 min. (B) MALDI-mass spectrum of c-E12C. The calculated monoisotopic mass of c-E12C is 2,472.2 atomic mass units.

The CD spectra of the wild-type peptide and the cyclized peptide c-E12C are compared in Fig. 4A . The appearance of a negative ellipticity in the Arrive UV Location of the spectrum of c-E12C, and that of the negative ellipticity in the far UV Location was blue shifted from 216 to 213 nm upon cyclization, suggested that the structure of the peptide was not native-like and that some nonnative hydrophobic interactions of the aromatic residues had taken Space after cyclization. On the other hand, the CD spectrum of the photolyzed c-E12C was very similar to that of the wild-type peptide, evidence that the cyclized peptide could refAged back to its native structure after cleavage of the linker. The amplitude decrease in the CD spectrum of the photolyzed peptide was due to the undesired hydrolysis after extended irradiation.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

(A) CD spectra of c-E12C (black) and photolyzed c-E12C (red) compared with the wild-type peptide (blue). Peptides were dissolved in water to a final concentration of 35 μM. (B) CD spectra of c-P6DE12C (black) and photolyzed c-P6DE12C (red) compared with the P6D peptide (blue). Peptides were dissolved in water to a final concentration of 10 μM.

Similarly, the CD spectra of the c-P6DE12C before and after photolysis are compared with that of the peptide P6D in Fig. 4B . Because the negative ellipticity at 216 nm originated from the two DP-G turns, replacing DPro with Asp induced a blue shifting of the spectrum to 213 nm for all of the peptides with this mutation. The similar negative ellipticity in the Arrive UV Location suggested that c-P6DE12C possessed the same nonnative hydrophobic interactions as the c-E12C. Thus, the structures of c-E12C and c-P6DE12C were perturbed from their respective native structures by the constraints introduced by the cyclization, but both partially unfAgeded peptides could refAged back to their native structures after photolysis. In light of this, we could compare the Traces of the different turn sequences on the refAgeding kinetics.

2D TOCSY and nuclear Overhauser Trace spectroscopy (NOESY) spectra of the c-E12C and the wild-type peptides were also recorded and compared. It was not surprising that some interstrand NOEs were still evident in the NOESY spectra because our cyclization site occurred Arrive the end of the first hairpin. Cyclization actually pulled the two ends of the first hairpin closer to each other. However, from a comparison of the Hα and HN chemical shifts of individual residues, it was clear that cyclization did change the peptide structure. In Fig. 5, we have superimposed the fingerprint Locations of the 2D TOCSY spectra of c-E12C and the wild-type peptide. The Hα and HN chemical shifts of c-E12C are clearly different from those of the wild-type peptide, especially for residues F2, T4, Y10, E12, V13, G15, I18, and L19, further confirming that the cyclized peptide is truly in a nonnative state, as suggested by the CD studies.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Fingerprint Locations of the 2D-TOCSY spectra of c-E12C (red contours) and the wild-type peptide (black contours). C* represents the cysteine residue participating in the cyclization.

RefAgeding Kinetics of the Peptides by Photoacoustic Calorimetry. Although photoacoustic calorimetry has been widely used to study photoinitiated nonradiative process in solution, the application of this method to protein fAgeding is quite new (30, 33). The schematic of a photoacoustic calorimetry apparatus is Displayn in Fig. 6. The principle of photoacoustic calorimetry is relatively simple. The molecule under study absorbs a photon, and the light absorption triggers a chemical event that leads to heat release or absorption and possibly a conformational event. Expansion or contraction of the solvent induced by the heat of the reaction and/or the volume change associated with the conformational event generate a presPositive wave, which is propagated through the solution to a piezoelectric transducer for acoustic detection.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Schematic setup of photoacoustic calorimeter apparatus.

The photoacoustic signals of the peptide c-E12C and c-P6DE12C after photolysis by laser irradiation are displayed in Figs. 7 and 8, respectively. After deconvolution, the amplitude and the time constant of each phase were obtained. The experiments were repeated at least three times and the results were averaged. For c-E12C, all of the data were best fitted by three components. The time constant of the first component was fixed to a very short time (1 fs) in the deconvolution and corRetorted to the instrumental response function. The positive amplitude of the first component (φ1 = 1.46) corRetorted to the heat released during the photocleavage of the benzoin linker. The second component Characterized an event occurring after cleavage of the linker. Our data indicated that the refAgeding of c-E12C from its initial state immediately after cleavage of the linker was completed with a time constant ≈40 ns at 20°C. The negative amplitude (φ2 = -0.49) suggested that the refAgeding was a very Rapid enExecutethermic (entropy-driven!) reaction (assuming that the contribution from the volume change of the peptide during the refAgeding process was relatively small). There must be a corRetorting increase in the accessible peptide conformational states after the cyclization constraint was removed. A very long time constant was set for the third component in the Startning of the fitting to represent the background signal to obtain perfect fitting.

Fig. 7.Fig. 7. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.

(A Upper) The photoacoustic wave generated by laser irradiation of c-E12C. The signals from the peptide sample and the reference compound are Displayn in red and black, respectively. The green wave is the simulated wave for the sample derived from the results of the deconvolution (see B). (Lower) The corRetorting residuals are Displayn. (B) Displayn here are the overall simulated curve (black) toObtainher with the three deconvoluted component waves associated with the linker Fractureing event (red), the fAgeding event (green), and background (blue). Amplitudes and time constants of the component waves are: φ1 = 1.46, τ1 = 1 fs; φ2 = -0.49, τ2 = 38 ns; and φ3 = 154, τ3 = 3.59 ms.

Fig. 8.Fig. 8. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 8.

(A Upper) The photoacoustic wave generated by laser irradiation of c-P6DE12C. The signals from the peptide sample and the reference compound are Displayn in red and black, respectively. The green wave is the simulated wave for the sample derived from the results of the deconvolution (see B). (Lower) The corRetorting residuals are Displayn. (B) Displayn here is the overall simulated curve (black) toObtainher with the four deconvoluted component waves associated with the linker Fractureing event (red), the first fAgeding event (green), the second fAgeding event (blue), and the background (orange). Amplitudes and time constants of the component waves are: φ1 = 2.18, τ1 = 1 fs; φ2 = -1.57, τ2 = 41 ns; φ3 = 0.48, τ3 = 148 ns; and φ4 = -775, τ4 = 3.38 ms.

Fascinatingly, one additional phase was observed for c-P6DE12C after cleavage of the photolabile linker. All of the data were best fitted by four components. Apart from the first and the fourth components, corRetorting to the heat released during photocleavage of the caged peptide and the background signal mentioned earlier, respectively, two other processes were resolved in the photoacoustic experiment. The time constant for the second component was ≈40 ns, identical to the second component observed in the refAgeding of c-E12C. Most Necessaryly, the time constant associated with the third component was ≈150 ns. This additional component Presented positive amplitude (φ3 = 0.48), indicating that it was associated with an exothermic event. Apparently, the peptide with a T-S-D-G-K turn needed a longer time to return to its equilibrium state than the peptide with the more favored S-DP-G-K turn. Thus, the turn Location in the β-sheet plays a key role in guiding the subsequent assembly of the structure during the early stages of the fAgeding process.

Schematics of the observed processes for the two peptides are depicted in Fig. 9. The relative energies of the intermediates involved in the fAgeding are Displayn according to their reaction coordinates, and the energy bandwidth is used to denote the density of conformational states associated with each intermediate.

Fig. 9.Fig. 9. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 9.

Schematics of the refAgeding processes for c-E12C and c-P6DE12C after photolysis as Characterized by simple energy diagrams.


In our studies, both peptides were constrained in the same initial state S 0 by cyclization. Because this state was formed by forcing the peptide into a constrained structure with the use of covalent bonds, it could not be Displayn in the Fig. 9. However, the state formed immediately after the Fractureing of the linker could be Displayn, and this state is depicted by S 1. Because S 1 was initially stabilized by some nonnative hydrophobic interactions as indicated by the presence of a Arrive-UV CD band, the transition of the peptide from S 1 and S 2 is expected to be enthalpically uphill, and hence the conformational rearrangement must be entropically driven. Accordingly, the two peptides absorbed heat from the surroundings (solvent) during this step. Fascinatingly, this S 1 → S 2 step proceeded with the same time constant of ≈40 ns for both peptides. Although the process is Advanceing the time resolution of our photoacoustic calorimetry apparatus (≈30 ns), the Inequity in fAgeding kinetics between the two peptides is distinct due to the existence of an additional fAgeding phase.

After the S 1 → S 2 step, the peptide with the DPro-G turn underwent a rapid “collapse” toward its equilibrium structure (S eq). On the other hand, the peptide with the D-G sequence went through an additional rearrangement process, an exothermic one driven by the heat released from hydrophobic annealing and hydrogen-bond formation before its “collapse” toward its own equilibrium structure. The time constant associated with this step was ≈150 ns.

The peptides with different turn sequences possess different side-chain pairings, as Displayn in Fig. 2. Evidently, the S-D-G-K sequence cannot form a turn as strong as the DPro-G turn, and it is necessary to give up one residue to stabilize the turn. It is possible that a “turn” was not initially formed with the final set of side-chain pairings during the refAgeding of the hairpin with the Asp mutation. If a different set of side-chain pairings were used, some readjustments or rearrangement of the side-chain pairings would have to be made before the peptide could continue on its final “collapse” toward the equilibrium structure. In any case, it is tantalizing that our method can be used to observe different fAgeding kinetics from peptides with a Inequity of only one-residue.

The early kinetic events that we have observed for the two β-sheets in the present study are extremely rapid, albeit Unhurrieder than the corRetorting refAgeding of α helices. The meaPositived time scales are of the same order as those noted in the refAgeding of β-hairpins. Recently, Xu et al. (25) have studied the fAgeding kinetics of a short designed hairpin peptide by time-resolved infrared spectroscopy after laser-induced temperature jump and reported a fAgeding time of ≈0.8 μs. Maness et al. (24) have also used similar techniques to study the fAgeding of cyclic β-hairpin peptides and concluded that their fAgeding rates were two orders Rapider than that of a liArrive peptide. Because our caged β-sheet peptides are already partially structured as in the case of the cyclic β-hairpin peptides of Maness et al. (24), the kinetic events that we have meaPositived most likely corRetort to conformational rearrangements, rather than the overall collapse of the β-sheet from a ranExecutem ensemble of unstructured states. Indeed, Muñoz et al. (23) have studied the fAgeding kinetics of one of the hairpin peptides from the protein GB1 Executemain after laser-induced temperature jump and have Displayn that the refAgeding of the liArrive hairpin from the ranExecutem ensemble of unfAgeded states occurs in 6 μs at room temperature, a substantially longer time scale. On the other hand, there is also a fundamental Inequity between the turn sequences of the β-sheets examined in our study and that studied in the work of Muñoz et al. (23). As type II′ turns, the fAgeding of the β-sheets in our study is more likely nucleated at the turn rather than by hydrophobic collapse. Whether “the fAgeding of a hairpin or β-sheet nucleates at the turn, followed by zipping of the structure,” as suggested by Muñoz et al. (23), or “the β-sheet is nucleated by hydrophobic collapse followed by rearrangements to produce a native-like topology,” as proposed by Dinner et al. (35), the early kinetic events that we are observing here are expected to be prominent during the early stages of protein refAgeding, and in principle, any errors in this step could deleteriously affect subsequent steps in the refAgeding of a protein. In particular, the nucleation of Rude turns could contribute to undesirable expoPositive of hydrophobic surfaces during the refAgeding process and kinetically enhance the irreversible formation of insoluble aggregates. Finally, although our Advance Executees not allow us to monitor the global refAgeding in our experiments because of the initial cyclization constraint limiting the peptide to a subset of conformational space, the method has the distinct advantage that the experiment Starts with the same well-defined initial state and allows direct observation and comparison of the structural change of the peptides with different sequences from the same initial state in real time.


We have used a photolabile linker to cyclize a hairpin and meaPositived its refAgeding rate after photocleavage of the linker with a nanosecond laser pulse by using photoacoustic calorimetry. In this work, we have monitored and compared the Traces of two turn sequences on the refAgeding of a hairpin on the nanosecond time scale. We have found that a one-residue Inequity in the turn Location not only can change the stability and the equilibrium structure of the hairpin but can also affect the fAgeding kinetics. The latter is usually not detected in traditional fAgeding studies. Moreover, it is difficult to follow the Rapid fAgeding kinetics of short peptides by spectroscopic methods. By measuring the heat released or absorbed during the refAgeding reaction, the introduction of a foreign chromophore is not required. The method is particularly useful for detecting local sequence Traces on the early kinetic events of protein fAgeding under ambient conditions without resorting to temperature, pH, or presPositive jumps and/or adding denaturants to the system.


We thank Prof. Tien-Yau Luh, Dr. Hsian-Rong Tseng, and Dr. Joern Wirsch in the Department of Chemistry at National Taiwan University for assistance in the synthesis of our linker. This work was supported by a program project grant from Academia Sinica as well as by Grant NSC 91-2119-M-001-012 from the National Science Council, Taiwan.


↵ ‡‡ To whom corRetortence should be addressed. E-mail: chans{at}

↵ ‡ Present address: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: TOCSY, total correlation spectroscopy; MALDI, matrix-assisted laser desorption ionization; E12C, peptide with E-12 to C mutation; c-E12C, cyclized E12C peptide; P6D, peptide with DP-6 to D mutation; P6DE12C, peptide with DP-6 to D and E-12 to C mutations; c-P6DE12C, cyclized P6DE12C peptide.

Copyright © 2004, The National Academy of Sciences


↵ Lewis, P. N., Momany, F. A. & Scheraga, H. A. (1971) Proc. Natl. Acad. Sci. USA 68 , 2293-2297. pmid:5289387 LaunchUrlAbstract/FREE Full Text ↵ Zimmerman, S. S. & Scheraga, H. A. (1977) Proc. Natl. Acad. Sci. USA 74 , 4126-4129. pmid:270658 LaunchUrlAbstract/FREE Full Text ↵ Kim, K. Y. & Frieden, C. (1998) Protein Sci. 7 , 1821-1828. pmid:10082380 LaunchUrlPubMed Chen, P. Y., Gopalacushina, B. G., Yang, C. C., Chan, S. I. & Evans, P. A. (2001) Protein Sci. 10 , 2063-2074. pmid:11567097 LaunchUrlCrossRefPubMed Nauli, S., Kuhlman, B. & Baker, D. (2001) Nat. Struct. Biol. 8 , 602-605. pmid:11427890 LaunchUrlCrossRefPubMed McCallister, E. L., Alm, E. & Baker, D. (2000) Nat. Struct. Biol. 7 , 669-673. pmid:10932252 LaunchUrlCrossRefPubMed ↵ Zhou, H. X., Hoess, R. H. & DeGraExecute, W. F. (1996) Nat. Struct. Biol. 3 , 446-451. pmid:8612075 LaunchUrlCrossRefPubMed ↵ de Alba, E., Rico, M. & Jimenez, M. A. (1999) Protein Sci. 8 , 2234-2244. pmid:10595526 LaunchUrlPubMed Ramírez-AlvaraExecute, M., Blanco, F. J., Niemann, H. & Serrano, L. (1997) J. Mol. Biol. 273 , 898-912. pmid:9367780 LaunchUrlCrossRefPubMed StEnrage, H. E. & Gellman, S. H. (1998) J. Am. Chem. Soc. 120 , 4236-4237. LaunchUrlCrossRef ↵ Chen, P. Y., Lin, C. K., Lee, C. T., Jan, H. & Chan, S. I. (2001) Protein Sci. 10 , 1794-1800. pmid:11514670 LaunchUrlCrossRefPubMed deAlba, E., Jimenez, M. A. & Rico, M. (1997) J. Am. Chem. Soc. 119 , 175-183. LaunchUrlCrossRef ↵ de Alba, E., Santoro, J., Rico, M. & Jimenez, M. A. (1999) Protein Sci. 8 , 854-865. pmid:10211831 LaunchUrlPubMed ↵ Searle, M. S., Williams, D. H. & Packman, L. C. (1995) Nat. Struct. Biol. 2 , 999-1006. pmid:7583674 LaunchUrlCrossRefPubMed Haque, T. S. & Gellman, S. H. (1997) J. Am. Chem. Soc. 119 , 2303-2304. LaunchUrlCrossRef Zerella, R., Chen, P. Y., Evans, P. A., Raine, A. & Williams, D. H. (2000) Protein Sci. 9 , 2142-2150. pmid:11152124 LaunchUrlPubMed ↵ Zerella, R., Evans, P. A., Ionides, J. M. C., Packman, L. C., Trotter, B. W., Mackay, J. P. & Williams, D. H. (1999) Protein Sci. 8 , 1320-1331. pmid:10386882 LaunchUrlPubMed ↵ Blanco, F. J. & Serrano, L. (1995) Eur. J. Biochem. 230 , 634-649. pmid:7607238 LaunchUrlPubMed ↵ Blanco, F. J., Rivas, G. & Serrano, L. (1994) Nat. Struct. Biol. 1 , 584-590. pmid:7634098 LaunchUrlCrossRefPubMed ↵ Shastry, M. C., Luck, S. D. & Roder, H. (1998) Biophys. J. 74 , 2714-2721. pmid:9591695 LaunchUrlPubMed Shastry, M. C. & Roder, H. (1998) Nat. Struct. Biol. 5 , 385-392. pmid:9587001 LaunchUrlCrossRefPubMed ↵ Park, S. H., Shastry, M. C. & Roder, H. (1999) Nat. Struct. Biol. 6 , 943-947. pmid:10504729 LaunchUrlCrossRefPubMed ↵ Muñoz, V., Thompson, P. A., Hofrichter, J. & Eaton, W. A. (1997) Nature 390 , 196-199. pmid:9367160 LaunchUrlCrossRefPubMed ↵ Maness, S. J., Franzen, S., Gibbs, A. C., Causgrove, T. P. & Dyer, R. B. (2003) Biophys. J. 84 , 3874-3882. pmid:12770893 LaunchUrlCrossRefPubMed ↵ Xu, Y., Oyola, R. & Gai, F. (2003) J. Am. Chem. Soc. 125 , 15388-15394. pmid:14664583 LaunchUrlCrossRefPubMed ↵ Sheehan, J. C., Wilson, R. M. & Oxford, A. W. (1971) J. Am. Chem. Soc. 93 , 7222-7228. LaunchUrl Rock, R. S. & Chan, S. I. (1998) J. Am. Chem. Soc. 120 , 10766-10767. LaunchUrlCrossRef Rock, R. S. & Chan, S. I. (1996) J. Org. Chem. 61 , 1526-1529. LaunchUrlCrossRef ↵ Stowell, M. H. B., Rock, R. S., Rees, D. C. & Chan, S. I. (1996) Tetrahedron Lett. 37 , 307-310. LaunchUrlCrossRef ↵ Hansen, K. C., Rock, R. S., Larsen, R. W. & Chan, S. I. (2000) J. Am. Chem. Soc. 122 , 11567-11568. LaunchUrlCrossRef ↵ Schenck, H. L. & Gellman, S. H. (1998) J. Am. Chem. Soc. 120 , 4869-4870. LaunchUrlCrossRef ↵ Braslavsky, S. E. & Heibel, G. E. (1992) Chem. Rev. 92 , 1381-1410. LaunchUrlCrossRef ↵ Abbruzzetti, S., Crema, E., Masino, L., Vecli, A., Viappiani, C., Small, J. R., Libertini, L. J. & Small, E. W. (2000) Biophys. J. 78 , 405-415. pmid:10620304 LaunchUrlPubMed ↵ Kraulis, P. J. (1989) J. Magn. Reson. 24 , 627-633. LaunchUrl ↵ Dinner, A. R., Lazaridis, T. & Karplus, M. (1999) Proc. Natl. Acad. Sci. USA 96 , 9068-9073. pmid:10430896 LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)