Elsa C. Y. Yan

Multifunctional Hybrid Nanogel for Integration of Optical Glucose Sensing and Self-Regulated Insulin Release at Physiological pH

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.

Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A crucial 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 that 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.

Chiral sum frequency generation spectroscopy for characterizing protein secondary structures at interfaces.

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.

Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin

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 protein-coupled receptor activation.

Second harmonic generation from the surface of centrosymmetric particles in bulk solution

Second harmonic generation (SHG) is reported for the first time from the surfaces of centrosymmetric particles in bulk isotropic solution. Although SHG is generally described as electric dipole forbidden in centrosymmetric systems, we show that this requires the system to be centrosymmetric on length scales much less than the coherence length of the process. This condition is not satisfied for micron-size particles, and accordingly we have observed a strong SH signal from various particles of this length scale. This promising discovery provides a powerful spectroscopic method for the investigation of physical and chemical processes on the surfaces of microscopic centrosymmetric particles.

In Situ Misfolding of Human Islet Amyloid Polypeptide at Interfaces Probed by Vibrational Sum Frequency Generation

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.

Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

Effect of Cholesterol on Molecular Transport of Organic Cations across Liposome Bilayers Probed by Second Harmonic Generation

The effect of cholesterol on the molecular transport of an organic cation, malachite green (MG), across large unilamellar dioleolyphosphatidylglycerol (DOPG) liposome bilayers with 0-50 mol% cholesterol was studied by second harmonic generation (SHG). Because SHG is a surface-specific technique, it requires no labeled molecule, quencher, or shifting agent to distinguish the location of the solute molecules. An additional important feature of SHG is that it is sensitive only to the probe molecules bound to the liposome, whereas other methods can only differentiate between molecules that are outside and those inside the liposome. The transport kinetics of MG across the liposome bilayers was observed in real time, and the results show that cholesterol retards the rate of transport of MG across liposome bilayers. The rate was found to decrease by six times for 50 mol% cholesterol content compared with cholesterol-free liposomes. This demonstrates the applicability of SHG to investigation of the effect of liposome composition on the transport kinetics across the liposome bilayers.

Amphiphilic Adsorption of Human Islet Amyloid Polypeptide Aggregates to Lipid/Aqueous Interfaces

Many amyloid proteins misfold into β-sheet aggregates upon interacting with biomembranes at the onset of diseases, such as Parkinsons disease and type II diabetes. The molecular mechanisms triggering aggregation depend on the orientation of β-sheets at the cell membranes. However, understanding how β-sheets adsorb onto lipid/aqueous interfaces is challenging. Here, we combine chiral sum frequency generation (SFG) spectroscopy and ab initio quantum chemistry calculations based on a divide-and-conquer strategy to characterize the orientation of human islet amyloid polypeptides (hIAPPs) at lipid/aqueous interfaces. We show that the aggregates bind with β-strands oriented at 48° relative to the interface. This orientation reflects the amphiphilic properties of hIAPP β-sheet aggregates and suggests the potential disruptive effect on membrane integrity.

Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors.

Functional reconstitution of transmembrane proteins remains a significant barrier to their biochemical, biophysical, and structural characterization. Studies of seven-transmembrane G-protein coupled receptors (GPCRs) in vitro are particularly challenging because, ideally, they require access to the receptor on both sides of the membrane as well as within the plane of the membrane. However, understanding the structure and function of these receptors at the molecular level within a native-like environment will have a large impact both on basic knowledge of cell signaling and on pharmacological research. The goal of this article is to review the main classes of membrane mimics that have been, or could be, used for functional reconstitution of GPCRs. These include the use of micelles, bicelles, lipid vesicles, nanodiscs, lipidic cubic phases, and planar lipid membranes. Each of these approaches is evaluated with respect to its fundamental advantages and limitations and its applications in the field of GPCR research. This article is part of a Special Issue entitled: Membrane protein structure and function.