In situ and real-time characterization of protein secondary structures at interfaces is important in biological and bioengineering sciences, yet remains technically challenging. In this study, we used chiral sum frequency generation (SFG) spectroscopy to establish a set of vibrational optical markers for characterizing protein secondary structures at interfaces. We discovered that the N-H stretches along the peptide backbones of α-helices can be detected in chiral SFG spectra. We further observed that the chiral vibrational signatures of the N-H stretch together with the peptide amide I are unique to α-helix, β-sheet, and random coil at interfaces. Using these chiral vibrational signatures, we studied the aggregation of human islet amyloid polypeptide (hIAPP), which is implicated in type II diabetes. We observed in situ and in real time the misfolding of hIAPP from random coils to α-helices and then β-sheets upon interaction with a lipid-water interface. Our findings show that chiral SFG spectroscopy is a powerful tool to follow changes in protein conformations at interfaces and identify interfacial protein secondary structures that elude conventional techniques.

CONTENTS 1. Introduction 8471 2. Theoretical Background of Chiral SFG 8472 2.1. General Principles of SFG 8472 2.2. Effective Susceptibility of Chiral Surfaces 8473 2.3. Surface Susceptibility and Molecular Hyperpolarizability 8474 2.4. Chiral SFG Response: Bulk versus Interface 8476 3. Chiral SFG Experiments 8477 3.1. Polarization Settings for Chiral SFG Experiments 8477 3.2. Spectrometers for Chiral SFG Studies of Biomacromolecules 8478 3.3. Surface Platforms for Probing Biomacromolecules 8479 4. Structures of Biomacromolecules at Interfaces Probed by Chiral SFG 8480 4.1. Chiral Amide I Signals of Proteins or Peptides at Interfaces 8480 4.2. Chiral C−H Stretch Signals of DNA on Solid/ Water Interfaces 8480 4.3. Chiral N−H Stretch from Protein Backbone at Interfaces 8481 4.4. Chiral N−H Stretch and Amide I for Probing Secondary Structures at Interfaces 8482 4.5. Characterization of Various Vibrational Bands of Collagen 8484 4.6. Double Resonance for Detecting Chiral SFG Signal from Porphyrin J Aggregates 8484 5. Orientations of Biomacromolecules at Interfaces Probed by Chiral SFG 8485 5.1. Orientation of Antiparallel β-Sheet Structures at Interfaces 8485 5.2. Orientation of Parallel β-Sheet Structures at Interfaces 8486 6. Kinetics and Dynamics of Biomacromolecules at Interfaces Probed by Chiral SFG 8488 6.1. Kinetics of Protein Folding Probed by Chiral Amide I and N−H Stretch 8488 6.2. Kinetics of Proton Exchange in Protein Backbones Probed by Chiral N−H 8489 6.3. Kinetics of Protein Self-Assembly Probed by Chiral C−H Stretch of Protein Side Chains 8490 7. Calculations of Hyperpolarizability of Biomacromolecules 8491 7.1. Calculation of Hyperpolarizability of Biomacromolecules for Weak Vibrational Coupling 8491 7.2. Calculation of Hyperpolarizability of Biomacromolecules for Strong Vibrational Coupling 8492 7.3. Calculation of Hyperpolarizability by the ab Initio Quantum Chemistry Method 8492 7.4. Comparison of Calculation Methods for Hyperpolarizability of Biomacromolecules 8493 8. Summary and Outlook 8493 8.1. Summary 8493 8.2. Potential Applications 8493 8.3. Challenges and Outlooks 8494 Author Information 8495 Corresponding Author 8495 Notes 8495 Biographies 8495 References 8496 Note Added after ASAP Publication 8498

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The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A critical question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein-coupled receptor.

A new noninvasive method for determining the surface electrostatic potential and surface charge density of microscopic particles using second harmonic generation (SHG) is described. The surface electrostatic properties of 1.05 µm polystyrene sulfate spheres in aqueous solution and that of 0.22 µm oil droplets in aqueous emulsions are obtained. Comparisons of the surface potentials obtained from SHG with the zeta potential obtained from electrophoresis are in excellent agreement with theoretical predictions.

The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pKa compared with the native pigment. This result is most consistent with the assignment of Glu-181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where the counterion switch occurs by transferring a proton from Glu-181 to Glu-113 through an H-bond network formed primarily with residues on extracellular loop II (EII). The resulting reorganization of EII is then coupled to movements of helix III through a conserved disulfide bond (Cys110 -Cys187); this process may be a general element of G proteincoupled receptor activation.

Optical detection of glucose, high drug loading capacity, and self-regulated drug delivery are simultaneously possible using a multifunctional hybrid nanogel particle under a rational design in a colloid chemistry method. Such hybrid nanogels are made of Ag nanoparticle (NP) cores covered by a copolymer gel shell of poly(4-vinylphenylboronic acid-co-2-(dimethylamino)ethyl acrylate) [p(VPBA-DMAEA)]. The introduction of the glucose sensitive p(VPBA-DMAEA) gel shell onto Ag NPs makes the polymer-bound Ag NPs responsive to glucose. While the small sized Ag cores (10 +/- 3 nm) provide fluorescence as an optical code, the responsive polymer gel shell can adapt to a surrounding medium of different glucose concentrations over a clinically relevant range (0-30 mM), convert the disruptions in homeostasis of glucose level into optical signals, and regulate release of preloaded insulin. This shows a new proof-of-concept for diabetes treatment that exploits the properties from each building block of a multifunctional nano-object. The highly versatile multifunctional hybrid nanogels could potentially be used for simultaneous optical diagnosis, self-regulated therapy, and monitoring of the response to treatment.

Kinetic analysis of conformational changes of proteins at interfaces is crucial for understanding many biological processes at membrane surfaces. In this study, we demonstrate that surface-selective sum frequency generation (SFG) spectroscopy can be used to investigate kinetics of conformational changes of proteins at interfaces. We focus on an intrinsically disordered protein, human islet amyloid polypeptide (hIAPP) that is known to misfold into the beta-sheet structure upon interaction with membranes. Using the ssp polarization setting (s-polarized SFG, s-polarized visible, and p-polarized infrared), we observe changes in the amide I spectra of hIAPP at the air/water interface after addition of dipalmitoylphosphoglycerol (DPPG) that correspond to the lipid-induced changes in secondary structures. We also used the chiral-sensitive psp polarization setting to obtain amide I spectra and observed a gradual buildup of the chiral structures that display the vibrational characteristics of parallel beta-sheets. We speculate that the second-order chiral-optical response at the antisymmetric stretch frequency of parallel beta-sheet at 1622 cm(-1) could be a highly characteristic optical property of the beta-sheet aggregates not only for hIAPP, but possibly also for other amyloid proteins. Analyzing the achiral and chiral amide I spectra, we conclude that DPPG induces the misfolding of hIAPP from alpha-helical and random-coil structures to the parallel beta-sheet structure at the air/water interface. We propose that SFG could complement existing techniques in obtaining kinetic and structural information for probing structures and functions of proteins at interfaces.