In this paper, we systematically presented the orientation determination of protein helical secondary structures using vibrational spectroscopic methods, particularly the nonlinear Sum Frequency Generation (SFG) vibrational spectroscopy, along with linear vibrational spectroscopic techniques such as infrared spectroscopy and Raman scattering. SFG amide I signals can be collected using different polarization combinations of the input laser beams and output signal beam to measure the second order nonlinear optical susceptibility components of the helical amide I modes, which are related to their molecular hyperpolarizability elements through the orientation distribution of these helices. The molecular hyperpolarizability elements of amide I modes of a helix can be calculated based on the infrared transition dipole moment and Raman polarizability tensor of the helix; these quantities are determined by using the bond additivity model to sum over the individual infrared dipole transition moments and Raman polarizability tensors, respectively, of the peptide units (or the amino acid residues). The computed overall infrared transition dipole moment and Raman polarizability tensor of a helix can be validated by experimental data using polarized infrared and polarized Raman spectroscopy on samples with well-aligned helical structures. From the deduced SFG hyperpolarizability elements and measured SFG second order nonlinear susceptibility components, orientation information regarding helical structures can be determined. Even though such orientation information can also be measured using polarized infrared or polarized Raman amide I signals, SFG has a much lower detection limit, which can be used to study the orientation of a helix when its surface coverage is much lower than a monolayer. In addition, the combination of different vibrational spectroscopic techniques, e.g., SFG and Attenuated Total Reflectance – Fourier Transform Infrared spectroscopy, provides more measured parameters for orientation determination, aiding in the deduction of more complicated orientation distributions. In this paper, we discussed two types of helices: the α-helix and 3–10 helix. However, the orientation determination method presented here is general, and thus can be applied to study other helices as well. The calculations of SFG amide I hyperpolarizability components for α-helical and 3–10 helical structures with different chain lengths have also been performed. It was found that when the helices reach a certain length, the number of peptide units in the helix should not alter the data analysis substantially. It was shown in the calculation, however, that when the helix chain is short, the SFG hyperpolarizability component ratios can vary substantially when the chain length is changed. Because 3–10 helical structures can be quite short in proteins, the orientation determination for a short 3–10 helix needs to take into account the number of peptide units in the helix.

Structural information such as orientations of interfacial proteins and peptides is important for understanding properties and functions of such biological molecules, which play crucial roles in biological applications and processes such as antimicrobial selectivity, membrane protein activity, biocompatibility, and biosensing performance. The α-helical and β-sheet structures are the most widely encountered secondary structures in peptides and proteins. In this paper, for the first time, a method to quantify the orientation of the interfacial β-sheet structure using a combined Attenuated Total Reflectance Fourier Transformation Infrared Spectroscopic (ATR-FTIR) and Sum Frequency Generation (SFG) vibrational spectroscopic study was developed. As an illustration of the methodology, the orientation of tachyplesin I, a 17-amino acid peptide with an anti-parallel β-sheet, adsorbed to polymer surfaces as well as associated with a lipid bilayer was determined using the regular and chiral SFG spectra, together with polarized ATR-FTIR amide I signals. Both the tilt angle (θ) and the twist angle (ψ) of the β-sheet at interfaces are determined. The developed method in this paper can be used to obtain in situ structural information of β-sheet components in complex molecules. The combination of this method and the existing methodology that is currently used to investigate α-helical structures will greatly broaden the application of optical spectroscopy in physical chemistry, biochemistry, biophysics, and structural biology.

We have designed and developed a novel sensor that reports the presence of specific nucleic acids in solutions, based on photon upconverting particles. The significantly high signal-to-noise ratio of photon upconverting particles leads to high sensitivity of the sensor. The sensor does not suffer from photobleaching. It also displays high specificity and self-calibrating capability. We expect nucleotide sensors of this type to be effective for applications in both DNA/RNA detection and protein-DNA/RNA interaction studies.

Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the nonglycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by ␤-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Gal␤1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Gal␤1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAc␤1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAc␤1-3(GlcA␤1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcAextended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.

In this paper, we investigated the molecular interactions of Magainin 2 with model cell membranes using Sum Frequency Generation (SFG) vibrational spectroscopy and Attenuated Total Reflectance -Fourier Transform Infrared spectroscopy (ATR-FTIR). Symmetric 1-Palmitoyl-2-Oleoyl-snGlycero-3-[Phospho-rac-(1-glycerol)] (POPG) and 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) bilayers, which model the bacterial and mammalian cell membranes respectively, were used in the studies. It was observed by SFG that Magainin 2 orients relatively parallel to the POPG lipid bilayer surface at low solution concentrations, around 200 nM. When increasing the Magainin 2 concentration to 800 nM, both SFG and ATR-FTIR results indicate that Magainin 2 molecules insert into the POPG bilayer and adopt a transmembrane orientation with an angle of about 20 degrees from the POPG bilayer normal. For the POPC bilayer, even at a much higher peptide concentration of 2.0 µM, no ATR-FTIR signal was detected. For this concentration on POPC, SFG studies indicated that Magainin 2 molecules adopt an orientation nearly parallel to the bilayer surface, with an orientation angle of 75 degrees from the surface normal. This shows that SFG has a much better detection limit than ATR-FTIR and can therefore be applied to study interfacial molecules with much lower surface coverage. This Magainin 2 orientation study and further investigation of the lipid bilayer SFG signals support the proposed toroidal pore model for the antimicrobial activity of Magainin 2.