Skip Williams

The helix is a common secondary structural motif found in proteins, and the mechanism of helix-coil interconversion is key to understanding the protein-folding problem. We report the observation of the fast kinetics (nanosecond to millisecond) of helix melting in a small 21-residue alanine-based peptide. The unfolding reaction is initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The model peptide exhibits fast unfolding kinetics with a time constant of 160 +/- 60 ns at 28 degrees C in response to a laser-induced temperature jump of 18 degrees C which is completed within 20 ns. Using the unfolding time and the measured helix-coil equilibrium constant of the model peptide, a folding rate constant of approximately 6 x 10(7) s-1 (t1/2 = 16 ns) can be inferred for the helix formation reaction at 28 degrees C. These results demonstrate that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.

We report the fast relaxation dynamics of "native" apomyoglobin (pH 5.3) following a 10-ns, laser-induced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales (nu = 1633 cm(-1),tau = 48 ns; nu = 1650 cm(-1),tau = 132 micros). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm(-1)) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm(-1)). Folding rate coefficients inferred from the observed relaxations at 60 degrees C are k(f)(solvated) = (7 to 20) x 10(6) s(-1) and k(f)(native) = 3.6 x 10(3) s(-1), respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60 degrees C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.

Diagrammatic perturbation theory combined with a spherical tensor treatment allows the degenerate four-wave mixing (DFWM) signal resulting from an isotropic molecular sample to be decomposed into a sum of three multipole moments in the weak-field (no saturation) limit. The zeroth moment gives the relative internal-state population contribution, the first moment the orientation contribution, and the second moment the alignment contribution to the DFWM spectra. This treatment makes explicit how the magnitude of the DFWM signal depends on the polarizations of the other three beams and the collisional relaxation caused by the environment. A general expression is derived for the DFWM signal for an arbitrary geometric configuration of the beams (arbitrary phase matching geometry). Under the assumption that the rates of collisional relaxation of the population, the orientation, and the alignment are the same, simple analytic expressions are found for the most commonly used experimental configurations, which should facilitate the practical analysis of DFWM spectra.

The degenerate four-wave-mixing (DFWM) signal is said to be saturated when the population difference of the two levels involved in the resonant transition oscillates with a rate (Rabi frequency) greater than the relaxation and dephasing rates. The field intensity at which this occurs is referred to as the DFWM saturation intensity. We find that the DFWM saturation behavior predicted by nondegenerate two-level models is in close agreement with the observed power dependence of (0,0) band transitions of the CH A 2Δ−X 2Π system. Furthermore, when the linear polarization states of the excitation fields are varied, the saturation intensity does not change significantly. In contrast, large differences in the DFWM signals are observed as a function of input field polarization and rotational branch. These differences are nearly independent of laser intensity. The DFWM signal differences are rationalized using the diagrammatic perturbation theory (DPT) expressions described in the preceding paper. We find that the DPT expressions are accurate to 10%–30% at saturating laser intensities. The important aspects of the reduction of saturated DFWM signals to relative internal-state distributions are outlined in environments where population relaxation and dephasing events are dominated by collisions, and a rotational temperature analysis is presented of the CH radical in an atmospheric-pressure oxyacetylene flame. Rotational temperatures determined using saturated DFWM are estimated to be accurate to 5%.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetection of trace species in hostile environments using degenerate four-wave mixing: methylidyne radical (CH) in an atmospheric-pressure flameSkip Williams, David S. Green, Srinivasan Sethuraman, and Richard N. ZareCite this: J. Am. Chem. Soc. 1992, 114, 23, 9122–9130Publication Date (Print):November 1, 1992Publication History Published online1 May 2002Published inissue 1 November 1992https://doi.org/10.1021/ja00049a053RIGHTS & PERMISSIONSArticle Views142Altmetric-Citations58LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alertsclose Get e-Alerts