Fluorescence-based methods to study rapid dynamics and conformational flexibility in peptides

Intramolecular collision of polypeptides is the primary step in protein folding, the dynamics of which is of importance for understanding this fascinating topic. In this thesis the rapid dynamics and flexibility of several sets of peptides were experimentally investigated with a fluorescence-based method, where the long-lived, hydrophilic fluorophore, 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO), was employed, which can be selectively and efficiently quenched by tryptophan (Trp) through contact. An asparagine derivative, Fmoc-DBO, was synthesized and applied to standard solid-phase peptide synthesis to obtain DBO/Trp-labeled peptides. The end-to-end collision rates can then be directly related to the intramolecular quenching of DBO by Trp. [Hudgins, R. R.; Huang, F.; Gramlich, G.; Nau, W. M. J. Am. Chem. Soc. 2002, 124, 556-564 (Appendix I); Nau, W. M.; Huang, F.; Wang, X.; Bakirci, H.; Gramlich, G.; Marquez, C. Chimia 2003, 57, 161-167 (Appendix III); Marquez, C.; Huang, F.; Nau, W. M. IEEE Trans. Nanobiosci. 2004, 3, 39-45 (Appendix V)] This method has been further improved by establishing a dual quencher system, i.e., tyrosine (Tyr) was employed as an additional quencher, which can react with DBO upon contact but with a lower efficiency than Trp. The combination of two probe/quencher pairs with different quenching efficiency as well as the theoretical results for intermolecular diffusion allows the extrapolation of the microscopic rate constants for formation and dissociation of the end-to-end encounter complex even in the absence of diffusion-controlled quenching. [Nau, W. M.; Huang, F.; Wang, X.; Bakirci, H.; Gramlich, G.; Marquez, C. Chimia 2003, 57, 161-167 (Appendix III); Huang, F.; Hudgins, R. R.; Nau, W. M. 2004, Submitted for publication (Appendix VI)] We first applied this fluorescence-based method to measure the end-to-end collision rate constants in flexible Gly-Ser peptides with varying length. The results suggest that the behavior of real peptides deviates significantly from that of the ideal chain model and the speed limit for protein folding should be faster than that reported previously. [Hudgins, R. R.; Huang, F.; Gramlich, G.; Nau, W. M. J. Am. Chem. Soc. 2002, 124, 556-564 (Appendix I)] We also investigated the end-to-end collision rates of another series of peptides composed of different types of amino acids in the backbone but with identical length. The experimental results have led to a conformational flexibility scale for amino acids in peptides and suggested that the flexibility of peptides is mainly determined by the atoms and groups in close proximity to the backbone, while the more remote atoms and groups have a smaller effect on the peptide dynamics due to their larger conformational space. [Huang, F.; Nau, W. M. Angew. Chem. Int. Ed. 2003, 42, 2269-2272 (Appendix II); Huang, F.; Nau, W. M. Res. Chem. Intermed. 2004, submitted for publication (Appendix VII)] Further investigations on peptides derived from the N-terminal b-hairpin of ubiquitin were also carried out. The end-to-end collision rates in these peptides showed significant dependence on the secondary structure, i.e., the turn segment is much more flexible than the strand segments, which supports a previous proposal that the b-turn is the initiator for the formation of the whole b-hairpin. Activation energies for end-to-end collision of these peptides showed a good agreement with the collision rate constants, which indicates that the activation energy may also be a measure of the flexibility of peptides although it is not as sensitive as the collision rate. [Huang, F.; Hudgins, R. R.; Nau, W. M. 2004, Submitted for publication (Appendix VI)] Additionally, to get more detailed structural information of our peptides and to reveal the underlying reasons for the deviation of the experimental length dependence of end-to-end collision rates from the theoretical prediction, intramolecular fluorescence resonance energy transfer (FRET) was applied as an independent approach to investigate the dynamics in peptide chains. Two energy donor/acceptor pairs with small Förster critical radius, where either naphthalene or Trp serves as energy donor and DBO as energy acceptor, were employed. Energy transfer between naphthalene and DBO was first investigated at a very short distance, where DBO and naphthalene were separated by dimethylsiloxy. It was found that the Dexter mechanism might dominate in this system due to the close proximity of donor and acceptor, the high flexibility of the tether, and the nonviscous solvent employed. [Pischel, U.; Huang, F.; Nau, W. M. Photochem. Photobiol. Sci. 2004, 3, 305-310 (Appendix IV)] However, when naphthalene and DBO were covalently attached to the opposite ends of peptides and studied in water, control experiments in the presence of cucurbit[7]uril as an encapsulating host suggested that FRET was the dominant mechanism, which allowed us to apply the FRET technique to recover the intramolecular end-to-end distance distribution and diffusion coefficient by means of global analysis. In the investigation with naphthalene/DBO energy donor/acceptor pair, slower diffusion coefficients in shorter chains were found for the series of flexible Gly-Ser peptides, suggesting that shorter chains may exhibit a larger internal friction limiting the conformational change. Additionally, the intramolecular energy transfer efficiency have been measured with the Trp/DBO pair and the effective average end-to-end distances were calculated, which provided a lower limit for the mean end-to-end distance of peptides for the global data analysis and offered a complementary approach to interpret the end-to-end collision rates determined with the same pair but based on a collision-induced quenching mechanism. [Huang, F.; Wang, X.; Haas, E.; Nau, W. M. 2004, In preparation (Appendix VIII)] The fluorescence-based method based on contact quenching mechanism has some other potential applications. It has potential to be applied for high-throughput screening of protease activity and to investigate the helix-coil transition in peptides

Probing DNA-Induced Colloidal Interactions and Dynamics with Scanning-Line Optical Tweezers

A promising route to forming novel nanoparticle-based materials is directed self-assembly, where the interactions among multiple species of suspended particles are intentionally designed to favor the self-assembly of a specific cluster arrangement or nanostructure. DNA provides a natural tool for directed particle assembly because DNA double helix formation is chemically specific — particles with short single-stranded DNA grafted on their surfaces will be bridged together only if those strands have complementary base sequences. Moreover, the temperature-dependent stability of such DNA bridges allows the resulting attraction to be modulated from negligibly weak to effectively irreversible over a convenient range of temperatures. Surprisingly, existing models for DNA-induced particle interactions are typically in error by more than an order of magnitude, which has hindered efforts to design complex temperature, sequence and time-dependent interactions needed for the most interesting applications. Here we report the first spatially resolved measurements of DNA-induced interactions between pairs of polystyrene microspheres at binding strengths comparable to those used in self-assembly experiments. The pair-interaction energies measured with our optical tweezers instrument can be modeled quantitatively with a conceptually straightforward and numerically tractable model, boding well for their application to direct self-assembly. In addition to understanding the equilibrium interactions between DNA-labeled particles, it is also important to consider the dynamics with which they bind to and unbind from one another. Here we demonstrate for the first time that carefully designed systems of DNA-functionalized particles exhibit effectively diffusion-limited binding, suggesting that these interactions are suitable to direct efficient self-assembly. We systematically explore the transition from diffusion-limited to reaction-limited binding by decreasing the DNA labeling density, and develop a simple dynamic model that is able to reproduce some of the anomalous kinetics observed in multivalent binding processes. Specifically, we find that when compounded, static disorder in the melting rate of single DNA duplexes gives rise to highly non-exponential lifetime distributions in multivalent binding. Together, our findings motivate a nanomaterial design approach where novel functional structures can be found computationally and then reliably realized in experiment

Energy Landscapes for Proteins: From Single Funnels to Multifunctional Systems

This report advances the hypothesis that multifunctional systems may be associated with multifunnel potential and free energy landscapes, with particular focus on biomolecules. It compares systems that exhibit single, double, and multiple competing structures, and contrasts multifunnel landscapes associated with misfolded amyloidogenic oligomers, which presumably do not arise as an evolutionary target. In this context, intrinsically disordered proteins could be considered intrinsically multifunctional molecules, associated with multifunnel landscapes. Potential energy landscape theory enables biomolecules to be treated in a common framework together with self‐organizing and multifunctional systems based on inorganic materials, atomic and molecular clusters, crystal polymorphs, and soft matter.epsr

Investigations of peptide structural stability in vacuo

Gas-phase analytical techniques provide very valuable tools for tackling the structural complexity of macromolecular structures such as those encountered in biological systems. Conformational dynamics of polypeptides and polypeptide assemblies underlie most biological functionalities, yet great difficulties arise when investigating such phenomena with the well-established techniques of X-ray crystallography and NMR. In areas such as these ion mobility interfaced with mass spectrometry (IMMS) and molecular modelling can make a significant contribution. During an IMMS experiment analyte ions drift in a chamber filled with an inert gas; measurement of the transport properties of analyte ions under the influence of a weak electric field can lead to determination of the orientationally-averaged collision cross-section of all resolved ionic species. A comparison with cross-sections estimated for model molecular geometries can lead to structural assignments. Thus IMMS can be used effectively to separate gas-phase ions based on their conformation. The drift tube employed in the experiments described herein is thermally regulated, which also enables the determination of collision cross-sections over a range of temperatures, and can provide a view of temperature-dependent conformational dynamics over the experimental (low microsecond) timescale. Studies described herein employ IMMS and a gamut of other MS-based techniques, solution spectroscopy and – importantly – molecular mechanics simulations to assess a) conformational stability of isolated peptide ions, with a focus on small model peptides and proteins, especially the Trp cage miniprotein; and b) structural characteristics of oligomeric aggregates of an amyloidogenic peptide. The results obtained serve to clarify the factors which dominate the intrinsic stability of non-covalent structure in isolated peptides and peptide assemblies. Strong electrostatic interactions are found to play a pivotal role in determining the conformations of isolated proteins. Secondary structures held together by hydrogen bonding, such as helices, are stable in the absence of solvent, however gas-phase protein structures display loss of their hydrophobic cores. The absence of a polar solvent, “self-solvation” is by far the most potent force influencing the gas-phase configuration of these systems. Geometries that are more compact than the folded state observed in solution are routinely detected, indicating the existence of intrinsically stable compact non-native states in globular proteins, illuminating the nature of proteins’ ‘unfolded’ states

Novel methods for manipulating ion types in the solution and gas phases for the structural analysis of biomolecules using Mass Spectrometry

Mass Spectrometry has become a valuable tool for the analysis of a variety of molecules, making it applicable to many fields. The advent of nanoelectrospray ionization (nESI) as a soft/low energy ionization technique has enabled the analysis of large, intact biomolecules. Most mass spectrometry experiments consist of three main steps: ionization, probe step(s), and mass analysis. The present work focuses on a variety of methods for altering ion types at various stages of the mass spectrometry experiment to affect ion fragmentation. Ion types can be manipulated in the solution/droplet phases using novel nESI emitters, generated from borosilicate theta capillaries. These nESI emitters enable the mixing of two solutions as they are sprayed into the mass spectrometer. This technique has been used to manipulate protein charge states (i.e. protein folding and unfolding) and to demonstrate peptide/protein analyte-reagent complex formation and covalent modification. This technique provides a simple and inexpensive method for manipulating ion types as the ions are generated during the electrospray process on the sub-millisecond timescale. These nESI emitters are also expanded to longer solution mixing times through the use of electroosmotic flow (EOF) between the two channels of the theta capillary prior to mass analysis. This work presents initial efforts to use theta capillaries to develop a lab-in-a-tip to provide for the manipulation of ion types on short timescales just prior to mass analysis. Additionally, ion types can be manipulated once the ions are in the gas-phase and trapped inside the mass spectrometer via ion/ion reactions. This work presents ion/ion reactions with reagents containing chromophores, which can be activated via ultraviolet photodissociation (UVPD) to generate radical peptide cations. Altering ion types in this way provides complementary sequence information upon collision induced dissociation (CID) when compared to CID of the even electron species. The McLuckey group is well known for work with ion/ion reactions to modify ion types and to conjugate biomolecules through covalent chemistry in the gas-phase. However, the kinetics and energetics of these reactions are not well known. This work will provide a method for measuring ion/ion reaction kinetics using dipolar DC CID (DDC-CID), which was previously developed in the McLuckey lab. Knowledge of gas-phase ion/ion reaction kinetics and energetics will provide insights for improving current ion/ion reaction efficiencies as well as for improving reagent design for future ion/ion reaction