Protein Folding and Self-Assembly

The folding of proteins and self-assembly of protein complexes arise from a complex interplay of non-covalent interactions involving the protein and water. Although local fluctuations on Ångstrom distance scales occur on femtosecond to picosecond time-scales, taken collectively they result in conformational changes of nanometer-size structures evolving on nanosecond to millisecond and longer time scales. Because of the complexity of protein structural evolution, we seek to identify and describe motion along collective coordinates, and answer questions about the process by which protein chains find specific contacts: What secondary and tertiary contacts are crucial to folding and binding? What is the role of water and hydrophobicity in guiding the collapse or assembly of structure? What are the intrinsic dynamical time-scales for folding?

Our approach uses 2D IR spectroscopy of the protein amide vibrations. The secondary structural sensitivity of amide I vibrations and the picosecond time resolution of vibrational spectroscopy makes 2D IR an excellent probe of protein dynamics. We have shown how the delocalized amide I vibration is sensitive to the number of hydrogen bond contacts in β sheets. Currently 2D IR experiments are used in combination with laser temperature jumps to probe unfolding on nanosecond to millisecond time scales. We also perform amide I experiments on peptides with isotope labels to probe site-specific structure and disorder. There is an ongoing effort to expand 2D IR spectroscopy to other protein vibrations, such as the amide II and II’ vibrations. A distinguishing quality of 2D IR is the availability of accurate structure-based models to calculate spectra from atomistic structures and MD simulations. This type of modeling plays an important part in our understanding of the spectroscopy, making predictions, and analyzing data.

Selected Reference:

Amide I Two-Dimensional Infrared Spectroscopy of Proteins Z. Ganim, H.S. Chung, A. Smith, L.P. DeFlores, K.C. Jones and A. Tokmakoff, Acc. Chem. Res. (2008).

(a) Amide I spectroscopy

The amide I protein vibration is primarily peptide carbonyl stretching. The amide I vibration is most sensitive to extended secondary structure due to inter- and intra-strand coupling and extended, periodic hydrogen bonding networks, which result in delocalization of the amide I mode over large regions of a protein. As a result, secondary structures may be identified based on their vibrational frequency. Amide I 2D IR spectroscopy provides a way of decomposing the congested amide I spectrum to help reveal underlying secondary structure content, size, and disorder. Detailed structure-based spectroscopic models exist that allow us to model amide I 2D IR spectra on the basis of a crystal structure or a molecular dynamics trajectory.

Selected References:

Spectral Signatures of Heterogeneous Protein Ensembles Revealed by MD Simulations of 2DIR Spectra Z. Ganim and A. Tokmakoff, Biophysical Journal, 91, 2636-2646 (2006).

Visualization and Characterization of the Infrared Active Amide I Vibrations of Proteins H. S. Chung, and A. Tokmakoff, J. Phys. Chem. B, 110, 2888-2898 (2006).

Two-dimensional infrared spectroscopy of antiparallel b-sheet secondary structure. N. Demirdöven, C. M. Cheatum, H. Chung, M. Khalil, J. Knoester and A. Tokmakoff, J. Am. Chem. Soc., 126, 7981(2004).

(b) Temperature-jump protein unfolding dynamics

Our efforts to describe the dynamics of protein folding are illustrated in recent studies of ubiquitin. We used amide I 2D IR and dispersed vibrational echo (DVE) experiments to characterize ubiquitin’s conformational changes on nanosecond to millisecond time scales accompanying unfolding induced by a T-jump. Using the analysis of spectra in conjunction with structure-based modeling of the unfolding process, we identified the unfolding pathway of ubiquitin based on the fast, probe-dependent downhill unfolding population: β-strands III-V unfold first with a 3 μs time constant, followed by an 80 μs loss of β-sheet character in the β-strand I,II hairpin, while the α-helix remains intact. Ongoing experiments seek to test the proposed unfolding mechanism with mutant studies, variable size temperature-jump experiments, and additional spectroscopic probes.

Selected References:

Transient 2D IR spectroscopy of ubiquitin unfolding dynamics H.S. Chung, Z. Ganim, K.C. Jones and A. Tokmakoff, PNAS, 36, 14237-14242 (2007).

Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin. H. Chung, M. Khalil, A. W. Smith, Z. Ganim, and A. Tokmakoff, PNAS, 102, 612-617 (2005).

(c) β-Hairpin folding

We study β-hairpin peptides as model systems to study different aspects of protein folding. β-hairpins fold to stabilize hydrophobic interactions, backbone hydrogen bonds, and β-turns, but simulations have yielded conflicting hypothesis for the mechanism. We seek to understand to what extent the kinetic zipper, hydrophobic collapse, or more complex heterogeneous pathways describe hairpin folding. Isotope labels are used to reveal site-specific information on the sequence of contact dissociation and water penetration between the β strands. With systematic studies of varying synthetic hairpins, we test the role of turn type and side-chain packing. Our T-jump studies of the β-hairpins PG-12 and TZ2 provide evidence for fraying, although TZ2 unfolding is distinctly heterogeneous.

Selected References:

Probing Local Structural Events in Beta-hairpin Unfolding with Transient Nonlinear Infrared Spectroscopy A. Smith and A. Tokmakoff, ACIE, 46(42), 7984-7987, (2007).

Residual Native Structure in a Thermally Denatured b-Hairpin A. W. Smith, H. S. Chung, Z. Ganim, and A. Tokmakoff, J. of Phys. Chem. B, 109, 17025-17027 (2005).

(e) Protein-water interactions

Current method development is aimed at gaining insight into the interactions between protein and water. One example of this work is our development of multimode 2D IR spectroscopy of the Amide I/II/II’ vibrations. This technique uses 2D IR in conjunction with H/D exchange measurements to correlate the secondary structure sensitivity of amide I vibrations with the sensitivity of amide II to solvent exposure. A protonated protein is placed in deuterated water which induces an H/D exchange of amide protons. The protonated Amide II vibration of the exchanging sight red shifts upon deuteration (Amide II’). By observing the 2D IR cross peak of Amide II and II’ with Amide I it is possible to map information on water accessibility which is provided by Amide II and II’ onto the secondary structure information provided by Amide I.

Selected Reference:

Water Penetration into Protein Secondary Structure Revealed by Hydrogen-Deuterium Exchange Two-Dimensional Infrared Spectroscopy L.P. DeFlores and A. Tokmakoff, J. Am. Chem. Soc. 128(51) 16520-16521, (2006).