β-Peptide foldamers offer attractive frameworks for examining the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding-unfolding kinetics of a β-peptide, Acid-1Y,1 which folds in aqueous solution into an octameric bundle of peptides in a conformation known as the 14-helix. Acid-1Y is comprised exclusively of β-amino acids, which differ from α-amino acids by the addition of a single methylene into the backbone. We aim to understand how the additional degree of freedom and increased backbone flexibility in the β-amino acid affect folding dynamics and to measure folding rates of this octameric β-peptide. Previously, we found that the T-jump induced relaxation kinetics of a monomeric β-peptide that forms a monomeric 14-helix occurred on the nanosecond time scale2 and were noticeably slower than a similar alanine-based α-helical peptide.3 Additionally, in comparison to similar α-helices, the relaxation rates showed a weaker dependence on temperature. Here, we find that the T-jump induced relaxation kinetics of the octameric β-peptide occurs on an even slower time scale (minutes) and the unfolding relaxation rates show a large dependence on temperature. These differences indicate that folding energy landscapes of β-peptide secondary and quaternary structure are markedly distinct from one another and also from their α-helical counterparts.

Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10-15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T_m ≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.

Because of its importance in viral replication, the M2 proton channel of the influenza A virus has been the focus of many studies. Although we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single-molecule fluorescence method to assess the dynamics of the inter-helical channel motion of both full-length M2 and the transmembrane domain of M2. The rate of this motion depends not only on the identity of the channel and membrane composition but also on the pH in a sigmoidal manner. For the full-length M2 channel, the rate is increased from approximately 190 μs^-1 at high pH to approximately 80 μs^-1 at low pH, with a transition midpoint at pH 6.1. Because the latter value is within the range reported for the conducting pK_a value of the His37 tetrad, we believe that this inter-helical motion accompanies proton conduction.

Recently, l-4-cyanotryptophan has been shown to be an efficient blue fluorescence emitter, with the potential to enable novel applications in biological spectroscopy and microscopy. However, lack of facile synthetic routes to this unnatural amino acid limits its wide use. Herein, we describe an expedient approach to synthesize Fmoc protected l-4-cyanotryptophan with high optical purity (>99%). Additionally, we test the utility of this blue fluorophore in imaging cell-membrane-bound peptides and in determining peptide-membrane binding constants.

Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measurable via folding kinetic studies. Specifically, we show that when a helix cross-linker, m-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C) but has little effect on protein stability. Thus, these results, in conjunction with those obtained with another cross-linked trp-cage and two uncross-linked variants, demonstrate the feasibility of using a nonperturbing cross-linker to help quantify the effect of internal friction. In addition, we estimate that a m-xylene cross-linker could lead to an increase in the roughness of the folding energy landscape by as much as 0.4-1.0k(B)T.

One of the fundamental events in protein folding is α-helix formation, which involves sequential development of a series of helical hydrogen bonds between the backbone C═O group of residues i and the -NH group of residues i + 4. While we now know a great deal about α-helix folding dynamics, a key question that remains to be answered is where the productive helical nucleation event occurs. Statistically, a helical nucleus (or the first helical hydrogen-bond) can form anywhere within the peptide sequence in question; however, the one that leads to productive folding may only form at a preferred location. This consideration is based on the fact that the α-helical structure is inherently asymmetric, due to the specific alignment of the helical hydrogen bonds. While this hypothesis is plausible, validating it is challenging because there is not an experimental observable that can be used to directly pinpoint the location of the productive nucleation process. Therefore, in this study we combine several techniques, including peptide cross-linking, laser-induced temperature-jump infrared spectroscopy, and molecular dynamics simulations, to tackle this challenge. Taken together, our experimental and simulation results support an α-helix folding mechanism wherein the productive nucleus is formed at the N-terminus, which propagates toward the C-terminal end of the peptide to yield the folded structure. In addition, our results show that incorporation of a cross-linker can lead to formation of differently folded conformations, underscoring the need for all-atom simulations to quantitatively assess the proposed cross-linking design.

Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA⁻protein interactions.

Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

The M2 proton channel of the influenza A virus has been the subject of extensive studies because of its critical role in viral replication. As such, we now know a great deal about its mechanism of action, especially how it selects and conducts protons in an asymmetric fashion. The conductance of this channel is tuned to conduct protons at a relatively low biologically useful rate, which allows acidification of the viral interior of a virus entrapped within an endosome, but not so great as to cause toxicity to the infected host cell prior to packaging of the virus. The dynamic, structural and chemical features that give rise to this tuning are not fully understood. Herein, we use a tryptophan (Trp) analog, 5-cyanotryptophan, and various methods, including linear and nonlinear infrared spectroscopies, static and time-resolved fluorescence techniques, and molecular dynamics simulations, to site-specifically interrogate the structure and hydration dynamics of the Trp41 gate in the transmembrane domain of the M2 proton channel. Our results suggest that the Trp41 sidechain adopts the t90 rotamer, the χ₂ dihedral angle of which undergoes an increase of approximately 35° upon changing the pH from 7.4 to 5.0. Furthermore, we find that Trp41 is situated in an environment lacking bulk-like water, and somewhat surprisingly, the water density and dynamics do not show a measurable difference between the high (7.4) and low (5.0) pH states. Since previous studies have shown that upon channel opening water flows into the cavity above the histidine tetrad (His37), the present finding thus provides evidence indicating that the lack of sufficient water molecules near Trp41 needed to establish a continuous hydrogen bonding network poses an additional energetic bottleneck for proton conduction.