Hauser, Karin


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Site-specific folding dynamics of isotopically labeled peptides studied by time-resolved infrared-spectroscopy

2009, Wu, Ling, Huang, Rong, Hauser, Karin, Krejtschi, Carsten, Keiderling, Timothy A.

Peptides with well-defined secondary structure are ideal model systems for study of protein folding dynamics for specific, unique structures. IR techniques provide the necessary time resolution as well as have structural sensitivity, which arises from coupling of sequential residues, normally evidenced as a splitting or frequency shift of the amide centered transitions. The amide I region, mainly the C¼O stretching vibrations of the polypeptide backbone, is the prime target band for secondary structure. Isotopic labeling of individual amide 13C¼O groups can induce site-specific frequency shifts and provide insight into local structure. A nanosecond laser is used to excite the solvent and induce a fast temperature jump (~10 C), and relaxation dynamics are probed with a diode laser tuned to selected, structurally sensitive wavenumbers across the amide I absorption. Site-specific dynamics have been monitored for the thermal unfolding of an isotopically labeled beta-hairpin peptide, a 12-mer tryptophan zipper peptide, which has a hydrophobic core formed by four Trp residues, by use of cross-strand coupled 13C¼O labeled variants [1]. Data for single labeled peptides provided a control. Mutants of this sequence with just two Trp residues were introduced to destabilize the hairpin selectively near the termini or near the turn. Differences in kinetic behavior have been found for the loss of beta-strand and the gain of disordered structure. The isotope-edited kinetics vary with labeling position along the hairpin backbone and the mutations show consistent patterns depending on position. Our data supports a multistate folding mechanism for this hairpin structure. Similarly obtained data for other model peptides provide useful basis for interpretation of the observations.

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Thermal unfolding of a beta hairpin studied with IR spectra enhanced by site-specific isotope labeling

2007, Huang, Rong, Krejtschi, Carsten, Hauser, Karin, Kim, Joohyun, Keiderling, Timothy A.

To model formation of beta-sheets in proteins it is essential to understand the interaction and dynamics between separate strands. The simplest model for such interactions are beta-hairpins, of which the TrpZip model of Cochran is one of the most stable in aqueous solution. We have modified the TrpZip2 sequence by substituting Ala on the 1,3, and 10 positions to destabilize the hairpin somewhat and to facilitate isotopic labeling. If C=O groups on opposite strands are labeled with 13C, their coupling can lead to distinctive amide I bands whose intensity, and sometimes frequency can reflect local cross-strand coupling, and thus hairpin formation at that position in the sequence. For a fully formed hairpin the A1A10 forms a labeled outer (14-atom) H-bonded ring and the A3K8 an inner one, while the A3A10 forms a central (10 atom) H-bonded ring. Equilibrium studies show the A1A10 to be less well formed (non-degenerate) than the A3K8, and the A3A10 has a different pattern with distinct disruptive effects on the 12C=O coupling. Laser excited T-jump measurements show fast relaxation kinetics that varies with wavelength probed and initial temperature. The differences in kinetics for different positions in the hairpin suggest a multistate process consistent with observations by others on related systems. Spectral simulations have been obtained using QM level force fields and structural results from extended MD calculations.

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Site-specific relaxation kinetics of a tryptophan zipper hairpin peptide using temperature-jump IR spectroscopy and isotopic labeling

2008-03-12, Hauser, Karin, Krejtschi, Carsten, Huang, Rong, Wu, Ling, Keiderling, Timothy A.

Two antiparallel â-strands connected by a turn make â-hairpins an ideal model system to analyze the interactions and dynamics of â-sheets. Site-specific conformational dynamics were studied by temperature-jump IR spectroscopy and isotopic labeling in a model based on the tryptophan zipper peptide, Trpzip2, developed by Cochran et al. (Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5578). The modified Trpzip2C peptides have nearly identical equilibrium spectral behavior as Trpzip2 showing that they also form wellcharacterized â-hairpin conformations in aqueous solution. Selective introduction of 13CdO groups on opposite strands lead to distinguishable cross-strand coupling of the labeled residues as monitored in the amide I¢ band. These frequency patterns reflect theoretical predictions, and the coupled 13CdO band loses intensity with increase in temperature and unfolding of the hairpin. Thermal relaxation kinetics were analyzed for unlabeled and cross-strand isotopically labeled variants. T-jumps of 10 °C induce relaxation times of a few microseconds that decrease with increase of the peptide temperature. Differences in kinetic behavior for the loss of â-strand and gain of disordered structure can be used to distinguish localized structure dynamics by comparison of nonlabeled and labeled amide I¢ components. Analysis of the data supports
multistate dynamic and equilibrium behavior, but because of this process it is not possible to clearly define a folding and unfolding rate. Nonetheless, site-specific relaxation kinetics could be seen to be consistent with a hydrophobic collapse hypothesis for hairpin folding.


Trpzip-Based Beta-Hairpin Equilibrium and Temperature Jump IR Studies Enhanced by Site-Specific Isotope Labeling

2008, Huang, Rong, Krejtschi, Carsten, Hauser, Karin, Keiderling, Timothy A.

Beta-hairpins may be the smallest folding units in a protein, and two antiparallel beta-strands connected by a turn make the simplest model system for analysis of the interactions and dynamics of betasheets. We have studied site-specific conformational dynamics by use of equilibrium and temperature-jump kinetic IR-spectroscopy with site-specific enhancement via isotopic labelling of the amide with 13C=O in isotopically labeled variants of a modification of Cochran’s 12-residue tryptophan zipper peptide, TrpZip2. Equilibrium measurements reflect decreased stability of the hairpin crossstrand H-bonds at the turn and the termini. Spectral analysis of single and doubly labeled species is used to determine specific coupling levels. 13C=O groups introduced at different amide positions lead to distinguishable cross-strand coupling of the labelled residues which is lost on unfolding. These labels have distinct frequency patterns and different thermal behaviors depending on their position in the hairpin and reflect the local structural variation along the strands. Relaxation kinetics upon laser-induced T-jumps of ~10 C have time constants of a few microsec that decrease with ncrease of the initial temperature of the peptide before the temperature jump. Analysis of the data supports a multistate folding process, consistent with the hydrophobic collapse hypothesis for hairpin folding, but it is not possible to clearly define a folding and unfolding rate.