Tryptophan as a Template for Development of Visible Fluorescent Amino Acids.

Most biological systems, at both molecular and cellular levels, are intrinsically complex, diverse, and nonfluorescent. Therefore, studying their structures, dynamics, and interactions via fluorescence-based methods requires incorporation of one or multiple external fluorophores that would not significantly affect any native property of the system in question. This requirement necessitates the development of a diverse set of fluorescence reporters that differ in chemical, physical, and photophysical properties. Herein, we offer our perspective on the need for, recent progress in, and future directions of developing tryptophan-based fluorescent amino acids.

Tuning the electronic transition energy of indole via substitution: application to identify tryptophan-based chromophores that absorb and emit visible light.

Fluorescent amino acids (FAAs) offer significant advantages over fluorescent proteins in applications where the fluorophore size needs to be limited or minimized. A long-sought goal in biological spectroscopy/microcopy is to develop visible FAAs by modifying the indole ring of tryptophan. Herein, we examine the absorption spectra of a library of 4-substituted indoles and find that the frequency of the absorption maximum correlates linearly with the global electrophilicity index of the substituent. This finding permits us to identify two promising candidates, 4-formyltryptophan (4CHO-Trp) and 4-nitrotryptophan (4NO2-Trp), both of which can be excited by visible light. Further fluorescence measurements indicate that while 4CHO-indole (and 4CHO-Trp) emits cyan fluorescence with a reasonably large quantum yield (ca. 0.22 in ethanol), 4NO2-indole is essentially non-fluorescent, suggesting that 4CHO-Trp (4NO2-Trp) could be useful as a fluorescence reporter (quencher). In addition, we present a simple method for synthesizing 4CHO-Trp.

A Scalable Synthesis of the Blue Fluorescent Amino Acid 4-Cyanotryptophan and the Fmoc Derivative: Utility Demonstrated with the Influenza M2 Peptide Tetramer.

A scalable synthesis of the Fmoc-protected blue fluorescent amino acid, l-4-cyanotryptophan (W4CN), that exploits an enantioselective phase transfer-catalyzed alkylation is reported. The red-shifted emission of water-exposed W4CN residues was leveraged to investigate the solvation state of tryptophan (Trp) residues within the influenza M2 proton channel. The correlation of the channel's conformation (i.e., open or closed) with the fluorescence spectrum of a mutated W4CN residue suggests that the channel's conformational state does not impact the hydration status of the Trp residues.

Assessing the Effect of Hofmeister Anions on the Hydrogen-Bonding Strength of Water via Nitrile Stretching Frequency Shift.

The temperature dependence of the peak frequency (νmax) of the C≡N stretching vibrational spectrum of a hydrogen-bonded C≡N species is known to be a qualitative measure of its hydrogen-bonding strength. Herein, we show that within a two-state framework, this dependence can be analyzed in a more quantitative manner to yield the enthalpy and entropy changes (ΔHHB and ΔSHB) for the corresponding hydrogen-bonding interactions. Using this method, we examine the effect of ten common anions on the strength of the hydrogen-bond(s) formed between water and the C≡N group of an unnatural amino acid, p-cyanophenylalanine (PheCN). We find that based on the ΔHHB values, these anions can be arranged in the following order: HPO42- > OAc- > F- > SO42- ≈ Cl- ≈ (H2O) ≈ ClO4- ≈ NO3- > Br- > SCN- ≈ I-, which differs from the corresponding Hofmeister series. Because PheCN has a relatively small size, the finding that anions having very different charge densities (e.g., SO42- and ClO4-) act similarly suggests that this ranking order is likely the result of specific ion effects. Since proteins contain different backbone and side-chain units, our results highlight the need to assess their individual contributions toward the overall Hofmeister effect in order to achieve a microscopic understanding of how ions affect the physical and chemical properties of such macromolecules. In addition, the analytical method described in the present study is applicable for analyzing the spectral evolution of any vibrational spectra composed of two highly overlapping bands.

4-Cyanoindole-based fluorophores for biological spectroscopy and microscopy.

Most biological molecules are intrinsically non- or weakly-fluorescent, hence requiring labeling with an external fluorophore(s) to be studied via fluorescence-based techniques. However, such labeling could perturb the native property of the system in question. One effective strategy to minimize such undesirable perturbation is to use fluorophores that are simple analogs of natural amino acids. In this chapter, we describe the synthesis and spectroscopic utility of two indole-based fluorophores, 4-cynaotryprophan (4CN-Trp) and 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS), with a focus on 4CN-Trp. This unnatural amino acid, which is only slightly larger than its natural counterpart, tryptophan (Trp), exhibits unique photophysical properties, making it a versatile fluorophore in biological spectroscopic and imaging applications. Through several specific examples, we highlight its broad utility in the study of various biological problems and processes.

Can glycine betaine denature proteins?

Glycine betaine (GB) is a naturally occurring osmolyte that has been widely recognized as a protein protectant. Since GB consists of a methylated ammonium moiety, it can engage in strong cation-π interactions with aromatic amino acid sidechains. We hypothesize that such specific binding interactions would allow GB to decrease the stability of proteins that are predominantly stabilized by a cluster of aromatic amino acids. To test this hypothesis, we investigate the effect of GB on the stability of two ß-hairpins (or mini-proteins) that contain such a cluster. We find that for both systems the stability of the folded state first decreases and then increases with increasing GB concentration. Such non-monotonic dependence not only confirms that GB can act as a protein denaturant, but also underscores the complex interplay between GB's stabilizing and destabilizing forces toward a given protein. While stabilizing osmolytes all have the tendency to be excluded from the protein surface which is the action underlying their stabilizing effect, our results suggest that in order to quantitatively assess the effect of GB on the stability of any given protein, specific cation-π binding interactions need to be explicitly considered. Moreover, our results show, consistent with other studies, that cation methylation can strengthen the respective cation-π interactions. Taken together, these findings provide new insight into the mechanism by which amino acid-based osmolytes interact with proteins.

Structural Destabilization of Azurin by Imidazolium Chloride Ionic Liquids in Aqueous Solution.

Alkyl imidazolium chloride ionic liquids (ILs) have been used for numerous biochemical applications. Their hydrophobicity can be tuned by changing the alkyl chain length, and longer-chain ILs can form micelles in aqueous solution. We have investigated the effects of imidazolium chloride ILs on the structure and stability of azurin, which is a very stable Cu2+ redox protein with both α-helix and ß-sheet domains. Temperature-dependent infrared (IR) and vibrational circular dichroism spectroscopy can provide secondary-structure-specific information about how the protein is affected, and temperature-jump transient IR measurements can quantify the IL-influenced unfolding dynamics. Using these techniques, we can quantify how azurin is destabilized by 1.0 M ILs in aqueous solution. The shorter, less hydrophobic ILs, 1-butyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium chloride likely interact with the α-helix domain and decrease protein melting temperature from 82 °C without IL to 55 °C and disturb the overall tertiary structure, resulting in a looser, more open shape. Thermodynamic analysis indicates that protein destabilization is due to increased unfolding entropy. 1-Octyl-3-methylimidazolium chloride [OMIM]Cl, which forms micelles in solution that may partially solvate the protein, has a more significant destabilizing effect, resulting in a melting temperature of 35 °C, larger unfolding entropy, and relaxation kinetics several orders of magnitude faster than with unperturbed azurin. The temperature-independence of the relaxation time constant suggests that in the presence of [OMIM]Cl, the protein folding potential energy surface has become very smooth.

4-Cyanoindole-2'-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics.

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.

Exposing the Nucleation Site in α-Helix Folding: A Joint Experimental and Simulation Study.

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.

Design of a Short Thermally Stable α-Helix Embedded in a Macrocycle.

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 Tm ≈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.

Region-Specific Double Denaturation of Human Serum Albumin: Combined Effects of Temperature and GnHCl on Structural and Dynamical Responses.

In this work, we have investigated the effects of denaturing agents, guanidine hydrochloride (GnHCl) and temperature, on the overall structure, domain-I, and domain-III of human serum albumin (HSA) using circular dichroism (CD) spectroscopy and steady-state, time-resolved fluorescence spectroscopy. We have tagged Cys-34 of HSA, located at domain-I, using N-(7-dimethylamino-4-methylcoumarin-3-yl)iodoacetamide and Tyr-411 of HSA, located at domain-III, using p-nitrophenyl coumarin ester, for this purpose. The CD spectroscopy studies reveal the overall denaturation of the protein. The denaturation follows the expected direction in which the protein is denatured with an increase in the concentration of GnHCl or temperature. The α-helicity of the native state of HSA was found to be 64.2%, and the minimum value of α-helicity was found to be 14.8% in the presence of 6 M GnHCl at room temperature. Steady-state emission studies were carried out on domain-I and domain-III of the protein using site-specific fluorescent tags. The degree of folding of the two domains at different combinations of temperature and GnHCl concentration was calculated and was found to follow a slightly different course of denaturation. Solvation dynamics was found to be quite different for these two domains. The domain-I of HSA has a maximum solvation time of 0.39 ns, and the solvation time tends to decrease with the action of either temperature or GnHCl. On the other hand, the domain-III of HSA showed a much higher solvation time (1.42 ns) and does not show any regular change at higher temperatures or in the presence of GnHCl. This difference could be attributed to the different microenvironment inside the protein cores of the two domains.

Elucidation of the local dynamics of domain-III of human serum albumin over the ps-µs time regime using a new fluorescent label.

The ps-µs dynamics of domain-III of human serum albumin (HSA) has been investigated using a new fluorescent marker selectively labeled to the Tyr-411 residue. The location of the marker has been confirmed using Förster resonance energy transfer (FRET) study. Steady state, time-resolved and single molecular level fluorescence techniques have been employed to understand the dynamics within the domain-III of HSA. It is found that solvent reorganization dynamics in domain-III is 1.7 times faster than that in domain-I. The timescale of the local rotational dynamics of domain-III is found to be 2.3 times faster than that of domain-I. Fluorescence correlation spectroscopic experiments reveal that domain-III of HSA has more conformational flexibility than domain-I. Together, the results deliver useful details of the local environment around the domain-III of HSA, which have not been explored earlier, mainly because of a lack of a suitable fluorescent marker for domain-III. The newly synthesized probe serves well as a site specific fluorescent marker for HSA, and can be used for further investigation of the ligand binding properties and enzymatic activity of domain-III of HSA.