University of Wyoming
Department of Chemistry

Kubelka Group
Protein Folding - Biomolecular Spectroscopy - Computational Chemistry

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Protein folding

Protein folding is one of the top 100 unsolved probems in science. We are focusing on small and structurally simple proteins, such as helix-turn-helix motifs, which are excellent models for obtaining new important insights into the secondary and tertiary structure formation in protein folding. Helix-turn-helix motifs are also the building blocks of all larger a-helical proteins. Detailed understanding of the folding mechanism of these simple motifs is a crucial step toward solving the protein folding problem in general.

To selectively probe structural changes within specific, local segments of the studied proteins during folding or unfolding, we use isotopically-edited infrared (IR) spectroscopy The site-specific resolution is achieved by incorporating 13C labeled amino acids, whose amide I (predominantly C=O stretch) vibrations are shifted in frequency and can be resolved in the protein IR spectra. To incorporate 13C labeled amino acids we synthesize the model proteins chamically by solid phase peptide synthesis (SPSS) methods. With chemical synthesis it is also straightforward to generate the model proteins with specific amino acid mutations. Combination of isotopic editing with site-directed mutational studies provides important insights into the effects of sequence-specific interactions on the folding process and the fundamental physical origins of the folding mechanism.

The isotopically-edited IR methodology is also very powerful for studying aggregation and fibril formation in proteins or model peptides, as well as for investigating peptide-membrane interactions. We are curently starting several projects in these areas.

Biomolecular spectroscopy

Spectroscopic methods are our main experimental tools for investigations of protein structural changes during folding or unfolding. We use mainly IR, but also ultra-violet circular dichroism (UVCD) and fluorescence spectroscopies. Recently, we have added Differential Scanning Calorimetry (DSC) to our repertoire of methods. In parallel to protein folding studies, we are constantly trying to gain better understanding of complex IR and CD spectra of proteins. We use a combintation of experimental studies on model compounds, such as N-methylacetamide (NMA), amino acids and small oligopeptides, with theoretical simulations of the spectra, predominantly based on density functional theory (DFT).

For example, solvent (water) can cause dramatic shifts in the IR band frequencies and intensities that are, furthermore, temperature dependent. Amino-acid side-chain vibrations can also interfere with the structurally sensitive amide backbone signals. Understanding these effects is critical for corect interpretation of the experimental IR data in terms of protein structural transitions.

Computational chemistry

Computer simulations are very valuable for understanding, prediction and interpretation of the experimental spectra. Simulations of the IR spectra, mainly based on quantum mechanical Density Functional Theory (DFT) methods, enable us to predict the expected 13C isotopic signals and to sort out the effects of conformational changes from those of the environment, such as solvent. Likewise, simulations of circular dichroism (CD) using time-dependent (TD) DFT, provide important insights into the effects of helix length, inter-helix interactions as well as solvent.

Other computational projects involve somulations of IR spectra of small molecules and CD of supramolecular assemblies and quantum dots, that are experimentally studied by other groups.

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