Nanosecond UV Resonance Raman Examination of Initial Steps in r-Helix Secondary Structure Evolution

The primary sequences of most proteins encode both the structure and the dynamics of the folding process. Unfortunately, it is still impossible to predict secondary and tertiary structures from primary sequences. This is because the complex folding dynamics involve structural evolution over several different time scales.1-4 Theoretical studies predict R-helix propagation on subnanosecond time scales, while nucleation of secondary structure motifs occurs on ns time scales and tertiary structure forms in the ms time scales. Recently, techniques such as fluorescence and IR spectroscopy have begun to probe protein folding dynamics in ns time scales.5-10 In this work we report the first application of ns transient UV resonance Raman spectroscopy (UVRS) to investigate the earliest events in protein structural evolution. We examine the thermal unfolding of the 21 amino acid R-helical peptide A5[AAARA]3A (AP), which occurs via two-state kinetics without any observable intermediates.5,6 We find that the unfolding rate constants show Arrhenius-type behavior with an apparent ∼7 kcal/mol barrier with a reciprocal rate constant of ∼200 ns at 37 °C. In contrast, the ∼1.1 μs folding rate constant shows a negative activation barrier. These results support recent protein folding landscape and funnel theories.1,2 UVRS excited in the 200 nm spectral region selectively probe protein secondary structure.11-13 This excitation selectively enhances amide vibrations because it is resonant with the amide backbone electronic transitions.14-16 These amide Raman bands sensitively depend on secondary structure. We recently determined the UVRS of the pure secondary structure Raman spectra (PSSRS) of the R-helix, -sheet and random coil motifs of proteins and demonstrated that these PSSRS can be used to quantitatively determine protein secondary structure.11 The static 204-nm UVRS of AP at high temperature are dominated by the Am I (1655 cm-1), Am II (1547 cm-1), CR-H bending (1382 cm-1), and Am III (1244 cm-1) bands (Figure 1a). The relative intensities and frequencies of these bands are very close to those of the random coil PSSRS.11 We conclude that high-temperature AP is essentially 100% random coil, which is also consistent with CD data. CD and the UVRS measurements indicate a decreasing R-helical content as the temperature increases. We calculated AP random coil and R-helix basis Raman spectra from the static spectra measured at several different temperatures (Figure 1a) by using CD measurements to determine the secondary structural composition. As a first approximation, we calculated the change in R-helical composition for each T-jump by measuring the average peak heights of the AIII, AII, and CR-H bending bands in the transient difference spectra. We compared these amplitudes to those of the steady-state temperature difference spectra. The UVRS changes between the sample at 4 °C and that measured 95 ns after a T-jump to 69 °C are identical to those expected upon partial unfolding of R-helical AP to the random coil17 (Figure 1b). Figure 1c shows that the UVRS changes depend on the probe delay time after a T-jump from 4 to 37 °C. The top * To whom correspondence should be addressed. (1) Dill, K. A.; Chan, H. S. Nat. Struct. Biol. 1997, 4, 10-19. (2) Chan, H. S.; Dill, K. A. Proteins: Struct. Funct. Genet. 1998, 30, 2-33. (3) Nymeyer, H.; Garcı́a, A. E.; Onuchic, J. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5921-5928. (4) Socci, N. D.; Onuchic, J. N.; Wolynes, P. G. J. Chem. Phys. 1996, 104, 5860-5868. (5) Williams, S.; Causgrove, T. P.; Gilmanshin, R.; Fang, K. S.; Callender, R. H.; Woodruff, W. H.; Dyer, R. B. Biochemistry 1996, 35, 691-697. (6) Thompson, P. A.; Eaton, W. A.; Hofrichter, J. Biochemistry 1997, 36, 9200-9210. (7) Gilmanshin, R.; Williams, S.; Callender, R. H.; Woodruff, W. H.; Dyer, R. B. Biochemistry 1997, 36, 15006-15012. (8) Lu, H. S. M.; Volk, M.; Kholodenko, Yu.; Gooding, E.; Hochstrasser, R. M.; DeGrado, W. F. J. Am. Chem. Soc. 1997, 119, 7173-7180. (9) Phillips, C. M.; Mizutani, Y.; Hochstrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7292-7296. (10) Ballew, R. M.; Sabelko, J.; Gruebele, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5759-5764. (11) Chi, Z.; Chen, X. G.; Holtz, J. S. W.; Asher, S. A. Biochemistry 1998, 37, 2854-2864. (12) Song, S.; Asher, S. A. J. Am. Chem. Soc. 1989, 111, 4295-4305. (13) Chi, Z.; Asher, S. A. Biochemistry 1998, 37, 2865-2872. (14) Li, P.; Chen, X. G.; Shulin, E.; Asher, S. A. J. Am. Chem. Soc. 1997, 119, 1116-1120. (15) Zhao, X.; Spiro, T. G. J. Raman Spectrosc. 1998, 29, 49-55. (16) Jordan, T.; Mukerji, I.; Wang, Y.; Spiro, T. G. J. Mol. Struct. 1996, 379, 51-64. (17) We Raman shifted the ∼3 ns, 1.06 μm fundamental of a Coherent Inc., Infinity YAG laser to 1.9 μm (1st H2 Stokes shift) to selectively heat the water solvent. The sample T-jump was independently measured using shifts in the ∼3400 cm-1 water Raman band. We probed the peptide structural evolution by exciting the UV Raman amide spectra with delayed 204 nm UV pulses generated by the 5th H2 anti-Stokes shifted frequency of the 3rd harmonic of the same YAG laser. The Raman instrumentation will be described in detail elsewhere. Raman scattering was measured from the surface of a 0.6-mm diameter thermostatically controlled sample solution stream. AP (95% purity) was prepared by the solid-phase peptide synthesis method. Figure 1. (a) Static 204-nm UV resonance Raman spectra of AP (15 mg/mL, pH 7 aqueous solution, no buffer) measured at several different temperatures. (b) Transient UVRS measured 95 ns after a T-jump from 4 to ∼69 °C. (c) Transient difference UVRS of AP initially at 4 °C at several different delay times after a T-jump of ∼33 °C. The static UVRS of AP at 4 °C is subtracted from each of the transient spectra. “Infinite” represents the difference between static UVRS measured at 37 and 4 °C. This static spectrum represents a difference spectrum at an infinite delay time. 4076 J. Am. Chem. Soc. 1999, 121, 4076-4077