Unusual Hydrophobic Property of Blue Fluorescent Amino Acid 4-Cyanotryptophan.

It is a common belief that the negative heat capacity change (Δ) associated with protein folding, which is a manifestation of the hydrophobic effect, results from a decrease in the solvent accessible hydrophobic surface area. Herein, we investigate the conformational energy landscape and dynamics of a tetrapeptide composed of two glycine and two 4-cyanotryptophan residues using time-resolved fluorescence spectroscopy, molecular dynamics simulations, and density functional theory calculations and find that, contrary to this expectation, the hydrophobic association of two 4-cyanotryptophan side chains leads to a positive Δ (approximately 543 J K mol). Furthermore, we find that promoting one of the 4-cyanotryptophans to its excited electronic state strengthens this self-association.

Conjugation of TLR7 and TLR7/8 agonists onto weak protein antigen via versatile oxime ligation for enhanced vaccine efficacy.

Protein-based subunit vaccines are weakly immunogenic, and developing self-adjuvanting vaccines with adjuvant conjugated to antigen is a promising approach for generating optimal immune responses. Here, we report a novel adjuvant-protein conjugate vaccine based on versatile oxime ligation technique. Firstly, the adjuvant properties of a series of TLR7 and TLR7/8 small molecule agonists in self-adjuvanting vaccines were systematically compared by coupling them to proteins in consistent ratio via p-carboxybenzaldehyde (p-CBA) for the first time.

Unnatural Amino Acids for Biological Spectroscopy and Microscopy.

Due to advances in methods for site-specific incorporation of unnatural amino acids (UAAs) into proteins, a large number of UAAs with tailored chemical and/or physical properties have been developed and used in a wide array of biological applications. In particular, UAAs with specific spectroscopic characteristics can be used as external reporters to produce additional signals, hence increasing the information content obtainable in protein spectroscopic and/or imaging measurements. In this Review, we summarize the progress in the past two decades in the development of such UAAs and their applications in biological spectroscopy and microscopy, with a focus on UAAs that can be used as site-specific vibrational, fluorescence, electron paramagnetic resonance (EPR), or nuclear magnetic resonance (NMR) probes.

Triple-Bond Vibrations: Emerging Applications in Energy and Biological Sciences.

Triple bonds, such as that formed between two carbon atoms (i.e., C≡C) or that formed between one carbon atom and one nitrogen atom (i.

C≡N Stretching Frequency as a Convenient Reporter of Charge Separation in Molecular Systems.

Previously, several studies have shown that, for a set of structurally related nitrile compounds, there could be a linear relationship between the total charge on the nitrile group () and its stretching frequency (ν). However, it is unclear whether the corresponding frequency and charge properties of structurally different nitrile compounds can be described by a single linear ν- relationship. Herein, we compute the magnitudes of a large number of nitrile-containing molecules whose ν values cover a spectral range of ca.

Photophysics of Two Indole-Based Cyan Fluorophores.

For the purpose of searching for new biological fluorophore, we assess the photophysical properties of two indole derivatives, 4-cyano-7-azaindole (4CN7AI) and 1-methyl-4-cyano-7-azaindole (1M4CN7AI), in a series of solvents. We find that (1) the absorption spectra of both derivatives are insensitive to solvents and are red-shifted from that of indole, having a maximum absorption wavelength of ca. 318 nm and a broad profile that extends beyond 370 nm; (2) both derivatives emit in the blue to green spectral range with a large Stokes shift, for example, in HO, the maximum emission wavelength of 4CN7AI (1M4CN7AI) is at ca.

Adjuvant-Free COVID-19 Vaccine with Glycoprotein Antigen Oxidized by Periodate Rapidly Elicits Potent Immune Responses.

Modification of antigens to improve their immunogenicity represents a promising direction for the development of protein vaccine. Here, we designed facilely prepared adjuvant-free vaccines in which the N-glycan of SARS-CoV-2 receptor-binding domain (RBD) glycoprotein was oxidized by sodium periodate. This strategy only minimally modifies the glycans and does not interfere with the epitope peptides.

4-Cyanotryptophan as a Sensitive Fluorescence Probe of Local Electric Field of Proteins.

Electrostatic interactions are key determinants of protein structure, dynamics, and function. Since protein electrostatics are nonuniform, assessment of the internal electric fields (EFs) of proteins requires spatial resolution at the amino acid residue level. In this regard, vibrational Stark spectroscopy, in conjunction with various unnatural amino acid-based vibrational probes, has become a common method for site-specific interrogation of protein EFs.

Photoenhancement of the C≡N Stretching Vibration Intensity of Aromatic Nitriles.

The C≡N stretching vibration is a versatile infrared (IR) reporter that is useful for a wide range of applications. Aiming to further expand its spectroscopic utility, herein, we show that, using 4-cyanoindole and 4-cyano-7-azaindole as examples, photoexcitation can significantly shift the frequency (ν) and enhance the molar extinction coefficient (ε) of this vibrational mode of aromatic nitriles and that, for these indole derivatives, the enhancement factor can reach 13. Moreover, we find that while solvent relaxation at the excited electronic state(s) always leads to an increase in ε, its effect on ν depends on the solute and the solvent.

Adjuvant-Protein Conjugate Vaccine with Built-In TLR7 Agonist on S1 Induces Potent Immunity against SARS-CoV-2 and Variants of Concern.

With the global pandemic of the new coronavirus disease (COVID-19), a safe, effective, and affordable mass-produced vaccine remains the current focus of research. Herein, we designed an adjuvant-protein conjugate vaccine candidate, in which the TLR7 agonist (TLR7a) was conjugated to S1 subunit of SARS-CoV-2 spike protein, and systematically compared the effect of different numbers of built-in TLR7a on the immune activity for the first time. As the number of built-in TLR7a increased, a bell-shaped reaction was observed in three TLR7a-S1 conjugates, with TLR7a(10)-S1 (with around 10 built-in adjuvant molecules on one S1 protein) eliciting a more potent immune response than TLR7a(2)-S1 and TLR7a(18)-S1.

A protein vaccine with Alum/c-GAMP/poly(I:C) rapidly boosts robust immunity against SARS-CoV-2 and variants of concern.

Adjuvants are important components in vaccines to increase the immunogenicity of proteins and induce optimal immunity. In this study, we designed a novel ternary adjuvant system Alum + c-GAMP + poly(I:C) with STING agonist 3,3'-c-GAMP (c-GAMP) and TLR3 agonist poly(I:C) co-adsorbed on the conventional adjuvant aluminum gel (Alum), and further constructed an S1 protein vaccine. Two doses of vaccination with the ternary adjuvant vaccine were sufficient to induce a balanced Th1/Th2 immune response and robust humoral and cellular immunity.

Analysis of serum metabolic profile by ultra-performance liquid chromatography-mass spectrometry for biomarkers discovery: application in a pilot study to discriminate patients with tuberculosis.

Background: Tuberculosis (TB) is a chronic wasting inflammatory disease characterized by multisystem involvement, which can cause metabolic derangements in afflicted patients. Metabolic signatures have been exploited in the study of several diseases. However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.

Reorientation of the "free OH" group in the top-most layer of air/water interface of sodium fluoride aqueous solution probed with sum-frequency generation vibrational spectroscopy.

Many experimental and theoretical studies have established the specific anion, as well as cation, effects on the hydrogen-bond structures at the air/water interface of electrolyte solutions. However, the ion effects on the top-most layer of the air/water interface, which is signified by the non-hydrogen-bonded so-called "free OH" group, have not been explicitly discussed or studied. In this report, we present the measurement of changes of the orientational angle of the "free OH" group at the air/water interface of the sodium fluoride (NaF) solutions at different concentrations using the interface selective sum-frequency generation vibrational spectroscopy (SFG-VS) in the ssp and ppp polarizations.

Mapping molecular orientation with phase sensitive vibrationally resonant sum-frequency generation microscopy.

We demonstrate a phase sensitive, vibrationally resonant sum-frequency generation (PSVR-SFG) microscope that combines high resolution, fast image acquisition speed, chemical selectivity, and phase sensitivity. Using the PSVR-SFG microscope, we generate amplitude and phase images of the second-order susceptibility of collagen I fibers in rat tail tendon tissue on resonance with the methylene vibrations of the protein. We find that the phase of the second-order susceptibility shows dependence on the effective polarity of the fibril bundles, revealing fibrous collagen domains of opposite orientations within the tissue.

Consistency in the sum frequency generation intensity and phase vibrational spectra of the air/neat water interface.

Substantial progress has been made in the quantitative understanding and interpretation of the hydrogen bonding and ordering structure of the air/water interface since the first sum-frequency generation vibrational spectroscopy (SFG-VS) measurement by Q. Du et al. in 1993 (Phys.

Spectroscopic evidence for the specific Na+ and K+ interactions with the hydrogen-bonded water molecules at the electrolyte aqueous solution surfaces.

Sum frequency generation vibrational spectra of the water molecules at the NaF and KF aqueous solution surfaces showed significantly different spectral features and different concentration dependence. This result is the first direct observation of the cation effects of the simple alkali cations, which have been believed to be depleted from the aqueous surface, on the hydrogen bonding structure of the water molecules at the electrolyte solution surfaces. These observations may provide important clues to understand the fundamental phenomenon of ions at the air/water interface.

Specific Na+ and K+ cation effects on the interfacial water molecules at the air/aqueous salt solution interfaces probed with nonresonant second harmonic generation.

Here we report on the polarization dependent nonresonant second harmonic generation (SHG) measurement of the interfacial water molecules at the aqueous solution of the following salts: NaF, NaCl, NaBr, KF, KCl, and KBr. Through quantitative polarization analysis of the SHG data, the orientational parameter D (D = /) value and the relative surface density of the interfacial water molecules at these aqueous solution surfaces were determined. From these results, we found that addition of each of the six salts caused an increase in the thickness of the interfacial water layer at the surfaces to a certain extent.

Increased interfacial thickness of the NaF, NaCl and NaBr salt aqueous solutions probed with non-resonant surface second harmonic generation (SHG).

Specific ion effects on the nonlinear optical response from the water molecules at the air/sodium halide solution interfaces are measured using non-resonant surface second harmonic generation (SHG). Procedures have been developed to monitor and remove the impurities in the salt solution samples to ensure measurement of small changes in the SHG signal. Quantitative polarization analysis of the measured SHG data indicated that the average orientation of the interfacial water molecules changed only slightly around 40 degrees with the increase of the bulk concentration of the three sodium halides, namely NaF, NaCl and NaBr, from that of the neat air/water interface.

Solvent effect on the film formation and the stability of the surface properties of poly(methyl methacrylate) end-capped with fluorinated units.

The surface structure and stability (the resistance to surface reconstruction) of end-capped poly(methyl methacrylate) films were greatly affected by the solvents used for film preparation. Films of end-capped PMMA with about four 2-perfluorooctylethyl methacrylate units cast with benzotrifluoride solution exhibited excellent stability and resistance to polar environments compared with those cast with cyclohexanone and toluene solutions. The observed difference in stability between these fluorinated surfaces is attributed to their surface microstructures formed during the film formation processes, which are closely related to the associative behavior of the end-capped PMMA in the solution.

Polarization and experimental configuration analyses of sum frequency generation vibrational spectra, structure, and orientational motion of the air/water interface.

Here we report a detailed study on spectroscopy, structure, and orientational distribution, as well as orientational motion, of water molecules at the air/water interface, investigated with sum frequency generation vibrational spectroscopy (SFG-VS). Quantitative polarization and experimental configuration analyses of the SFG data in different polarizations with four sets of experimental configurations can shed new light on our present understanding of the air/water interface. Firstly, we concluded that the orientational motion of the interfacial water molecules can only be in a limited angular range, instead of rapidly varying over a broad angular range in the vibrational relaxation time as suggested previously.