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In JoVE (1)
Other Publications (5)
Articles by Diana E. Schlamadinger in JoVE
Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy
Diana E. Schlamadinger, Judy E. Kim
Chemistry and Biochemistry, University of California San Diego - UCSD
This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.
Other articles by Diana E. Schlamadinger on PubMed
Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA
Biochemistry. Dec, 2008 | Pubmed ID: 18991402
Refolding curves of the integral membrane protein outer membrane protein A (OmpA) were measured to determine the conformational stabilities of this model system for membrane protein folding. Wild-type OmpA exhibits a free energy of unfolding (DeltaG degrees H2O) of 10.5 kcal/mol. Mutants, containing a single tryptophan residue at the native positions 7, 15, 57, 102, or 143, are less stable than wild-type OmpA, with DeltaG degrees H2O values of 6.7, 4.8, 2.4, 4.7, and 2.8 kcal/mol, respectively. The trend observed here is discussed in terms of noncovalent interactions, including aromatic interactions and hydrogen bonding. The effect of the soluble tail on the conformational stability of the transmembrane domain of OmpA was also investigated via truncated single-Trp mutants; DeltaG degrees H2O values for four of the five truncated mutants are greater by >2.7 kcal/mol relative to the full-length versions, suggesting that the absence of the soluble domain may destabilize the unfolded transmembrane domain. Finally, dynamic light scattering experiments were performed to measure the effects of urea and protein on vesicle size and stability. Urea concentrations greater than 1 M cause an increase in vesicle size, and these diameters are unaltered in the presence of protein. These dynamic light scattering results complement the fluorescence studies and illustrate the important effects of vesicle size on protein conformational stability.
Förster Resonance Energy Transfer and Conformational Stability of Proteins: An Advanced Biophysical Module for Physical Chemistry Students
Journal of Chemical Education. Sep, 2008 | Pubmed ID: 19756254
Protein folding is an exploding area of research in biophysics and physical chemistry. Here, we describe the integration of several techniques, including absorption spectroscopy, fluorescence spectroscopy, and Förster resonance energy transfer (FRET) measurements, to probe important topics in protein folding. Cytochrome c is used as a model protein; comparison of conformational stabilities ( ΔGH2O∘) measured via two chemical denaturants, urea and guanidinium hydrochloride, illustrate important concepts in protein folding and intermolecular interactions. In addition, the determination of intraprotein distances based upon the FRET pair Trp-59 and the heme group for unfolded states of cytochrome c highlights the evolution of the protein structure under unfolding conditions. Analysis and discussion of these results provide opportunities to gain in-depth understanding of models for protein folding while enhancing students' skills with optical techniques. Collectively, the combination of optical spectroscopy, rigorous quantitative analysis, and a focus on biophysics illustrates the significance of fundamental research at the growing intersection of chemistry, biology, and physics.
Hydrogen Bonding and Solvent Polarity Markers in the Uv Resonance Raman Spectrum of Tryptophan: Application to Membrane Proteins
The Journal of Physical Chemistry. B. Nov, 2009 | Pubmed ID: 19817473
Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-pi interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I1360/I1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters pi* and ETN. As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (approximately 1237 cm-1) intensity to that of the W9 (approximately 1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-pi interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-pi interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides.
Fluorescence and UV Resonance Raman Study of Peptide-vesicle Interactions of Human Cathelicidin LL-37 and Its F6W and F17W Mutants
Biochemistry. Dec, 2009 | Pubmed ID: 19894716
LL-37 is a broad-spectrum human antimicrobial peptide in the cathelicidin family. Potency assays in the form of minimal inhibitory concentration and vesicle leakage indicate that the single-tryptophan mutants, F6W and F17W, are as effective at killing bacteria and disrupting membranes as the native, tryptophan-free LL-37 peptide. Steady-state fluorescence and UV resonance Raman spectroscopy of F6W and F17W reveal molecular details of these tryptophan residues. The local environment polarity, hydrogen bond strength of the indole N-H moiety, and rotational freedom decrease for both F6W and F17W in the presence of carbonate ions relative to in pure distilled water; these results are consistent with burial of the hydrophobic region of alpha-helical LL-37 in oligomeric cores induced in the presence of carbonate ions. Differences in the spectroscopic properties of the carbonate-induced alpha-helical forms of F6W and F17W reflect the presence of a local lysine residue near F6W that makes the microenvironment of F6W more polar than that of F17W. In the presence of lipid vesicles, the mutants undergo additional loss of environment polarity, hydrogen bond strength, and rotational freedom. Quenching experiments utilizing brominated lipids reveal that the tryptophan residues in both mutants are essentially equidistant from the bilayer center and that bromines closer to the bilayer center, in the 9,10 positions, quench fluorescence more efficiently than those closer to the headgroups (6,7 positions). These results support carpeting or toroidal pore mechanisms of membrane disruption by LL-37 and demonstrate that the combination of tryptophan mutants and sensitive spectroscopic tools may provide important molecular clues about antimicrobial action.
Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Nov, 2011 | Pubmed ID: 21910205