Ronald Soong

Association of Highly Compact Type II Diabetes Related Islet Amyloid Polypeptide Intermediate Species at Physiological Temperature Revealed by Diffusion NMR Spectroscopy

Self-association of human islet amyloid polypeptide (hIAPP) is correlated with the development of type II diabetes by the disruption of cellular homeostasis in islet cells through the formation of membrane-active oligomers. The toxic species of hIAPP responsible for membrane damage has not been identified. In this study, we show by pulsed field gradient NMR spectroscopy that the monomeric form of the toxic, amyloidogenic human variant of IAPP (hIAPP) adopts a temperature dependent compact folded conformation that is absent in both the nontoxic and nonamyloidogenic rat variant of IAPP and absent in hIAPP at low temperatures, suggesting this compact form of monomeric hIAPP may be linked to its later aggregation and cytotoxicity. In addition to the monomeric form of hIAPP, a large oligomeric species greater than 100 nm in diameter is also present but does not trigger the nucleation-dependent aggregation of IAPP at 4 degrees C, indicating the large oligomeric species may be an off-pathway intermediate that has been predicted by kinetic models of IAPP fiber formation. Furthermore, analysis of the polydispersity of the calculated diffusion values indicates small oligomeric species of hIAPP are absent in agreement with a recent ultracentrifugation study. The absence of small oligomeric species in solution suggests the formation of small, well-defined ion channels by hIAPP may proceed by aggregation of monomeric IAPP on the membrane, rather than by the insertion of preformed structured oligomers from the solution state as has been proposed for other amyloidogenic proteins.

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Background: cytb5 modulates catalysis performed by cytsP450, in vivo and in vitro. Results: The structure of full-length cytb5 was solved by NMR, and the cytP450-binding site on cytb5 was identified by mutagenesis and NMR. Conclusion: A model of the cytb5-cytP450 complex is presented. Addition of a substrate strengthens the cytb5-cytP450 interaction. Significance: The cytb5-cytP450 complex structure will help unravel the mechanism by which cytb5 regulates catalysis by cytP450. Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.

When detergent meets bilayer: birth and coming of age of lipid bicelles.

Lipids spontaneously form bilayered structures when brought into an aqueous environment. This is the foundation in the architecture of biological cell membranes. However, lipid bilayers do not lend themselves easily to common biophysical studies; be it of the bilayer itself or of embedded membrane proteins. Detergents, on the other hand, form small aggregates known as micelles that readily solubilize membrane proteins and are well-suited for numerous biophysical methods. However, they are not excellent models of biological membranes as they may denature the structure of a protein and the curvature of the micelle may impose a non-native protein folding. When lipid and detergent meet in an aqueous environment, entities with wholly different properties are formed: lipid bicelles. Bicelles are made of patches of lipid bilayers that are either encircled or perforated by detergent ‘rims’. They combine the advantages of both components alone (micelle and lipid bilayer), namely being good models for a biological membrane and having advantageous properties for biophysical experiments. An additional advantage of certain bicelle preparations is their tendency to macroscopically align when brought into a magnetic field. This fact has been exploited not only in the highresolution structural and dynamics studies of membrane proteins, but also for globular proteins using nuclear magnetic resonance (NMR) experiments. Fig. 1 gives a graphical introduction to the two types of bicellar phases most commonly employed. At a high detergent concentration and low temperatures, isotropically tumbling disk-like aggregates are formed, the so-called isotropic bicelles (Fig. 1B). At a high lipid concentration and in certain temperature ranges, extended bilayered lamellae are formed that are perforated or delimited by detergent, and have the potential for magnetic alignment (Fig. 1D). Cryo-transmission electron microscopy (TEM) micrographs (A, C) of bicelles taken from the literature [1] are also included in Fig. 1. Fig. 1 Lipid bicelles are supramolecular aggregates that are formed when appropriate amounts of lipids and detergents are mixed in an aqueous environment. The size and phase of bicellar aggregates depend on the [lipid]:[detergent] ratio as well as on the temperature. ... Since their first description in 1988, the great potential of bicelles in the study of membrane proteins and proteins in general has been realized. A steady stream of remarkable insights and applications has emerged that is still growing in size. In the present contribution, we will give an introduction to the properties of lipid bicelle phases with an emphasis on NMR experimental measurements. In addition, we will discuss some of the most exciting recent applications of bicelles in the structural and dynamic studies of membrane proteins.

Site specific interaction of the polyphenol EGCG with the SEVI amyloid precursor peptide PAP(248-286).

Recently, a 39 amino acid peptide fragment from prostatic acid phosphatase has been isolated from seminal fluid that can enhance infectivity of the HIV virus by up to 4-5 orders of magnitude. PAP(248-286) is effective in enhancing HIV infectivity only when it is aggregated into amyloid fibers termed SEVI. The polyphenol EGCG (epigallocatechin-3-gallate) has been shown to disrupt both SEVI formation and HIV promotion by SEVI, but the mechanism by which it accomplishes this task is unknown. Here, we show that EGCG interacts specifically with the side chains of monomeric PAP(248-286) in two regions (K251-R257 and N269-I277) of primarily charged residues, particularly lysine. The specificity of interaction to these two sites is contrary to previous studies on the interaction of EGCG with other amyloidogenic proteins, which showed the nonspecific interaction of EGCG with exposed backbone sites of unfolded amyloidogenic proteins. This interaction is specific to EGCG as the related gallocatechin (GC) molecule, which shows greatly decreased antiamyloid activity, exhibits minimal interaction with monomeric PAP(248-286). The EGCG binding was shown to occur in two steps, with the initial formation of a weakly bound complex followed by a pH dependent formation of a tightly bound complex. Experiments in which the lysine residues of PAP(248-286) have been chemically modified suggest the tightly bound complex is created by Schiff-base formation with lysine residues. The results of this study could aid in the development of small molecule inhibitors of SEVI and other amyloid proteins.

Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy

In addition to providing a semipermeable barrier that protects a cell from harmful stimuli, lipid membranes occupy a central role in hosting a variety of biological processes, including cellular communications and membrane protein functions. Most importantly, protein-membrane interactions are implicated in a variety of diseases and therefore many analytical techniques were developed to study the basis of these interactions and their influence on the molecular architecture of the cell membrane. In this study, sum frequency generation (SFG) vibrational spectroscopy is used to investigate the spontaneous membrane insertion process of cytochrome b(5) and its mutants. Experimental results show a significant difference in the membrane insertion and orientation properties of these proteins, which can be correlated with their functional differences. In particular, our results correlate the nonfunctional property of a mutant cytochrome b(5) with its inability to insert into the lipid bilayer. The approach reported in this study could be used as a potential rapid screening tool in measuring the topology of membrane proteins as well as interactions of biomolecules with lipid bilayers in situ.

Comprehensive Analysis of Lipid Dynamics Variation with Lipid Composition and Hydration of Bicelles Using Nuclear Magnetic Resonance (NMR) Spectroscopy

Bicelles of various lipid/detergent ratios are commonly used in nuclear magnetic resonance (NMR) studies of membrane-associated molecules without the need to freeze the sample. While a decrease in the size (defined at a low temperature or by the q value) of a bicelle decreases its overall order parameter, the variation of lipid dynamics with a change in the lipid/detergent ratio is unknown. In this study, we report a thorough atomistic level analysis on the variation of lipid dynamics with the size and hydration level of bicelles composed of a phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and a detergent, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). Two-dimensional (2D) separated-local-field NMR experiments were performed on magnetically aligned bicelles to measure (1)H-(13)C dipolar couplings, which were used to determine order parameters at various (head-group, glycerol, and acyl chain) regions of lipids in the bilayer. From our analysis, we uncover the extreme sensitivity of the glycerol region to the motion of the bicelle, which can be attributed to the effect of viscosity because of an extensive network of hydrogen bonds. As such, the water-membrane interface region exhibits the highest order parameter values among all three regions of a lipid molecule. Our experimental results demonstrate that the laboratory-frame 2D proton-detected-local-field pulse sequence is well-suited for the accurate measurement of motionally averaged (or long-range) weak and multiple (1)H-(13)C dipolar couplings associated with a single carbon site at the natural abundance of (13)C nuclei.

Proton-evolved local-field solid-state NMR studies of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information.

Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented two-dimensional proton-evolved local-field (2D PELF) pulse sequences using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically aligned bicelles containing uniformly (15)N-labeled cytochrome b(5) are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF-based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.

INEPT-based separated-local-field NMR spectroscopy: a unique approach to elucidate side-chain dynamics of membrane-associated proteins.

Despite recent advances in NMR approaches for structural biology, determination of membrane protein dynamics in its native environment continues to be a monumental challenge, as most NMR structural studies of membrane proteins are commonly carried out either in micelles or in vesicle systems under frozen conditions. To overcome this difficulty, we propose a solid-state NMR technique that allows for the determination of side-chain dynamics from membrane proteins in lipid bilayers. This new technique, namely dipolar enhanced polarization transfer (DREPT), allows for a wide range of dipolar couplings to be encoded, providing high resolution and sensitivity for systems that undergo motional averaging such as that of amino acid side chains. NMR observables such as dipolar couplings and chemical shift anisotropy, which are highly sensitive to molecular motions, provide a direct way of probing protein dynamics over a wide range of time scales. Therefore, using an appropriate model, it is possible to determine side-chain dynamics and provide additional information on the topology and function of a membrane protein in its native environment.

The amyloidogenic SEVI precursor, PAP248-286, is highly unfolded in solution despite an underlying helical tendency

Amyloid fibers in human semen known as SEVI (semen-derived enhancer of viral infection) dramatically increase the infectivity of HIV and other enveloped viruses, which appears to be linked to the promotion of bridging interactions and the neutralization of electrostatic repulsion between the host and the viral cell membranes. The SEVI precursor PAP(248-286) is mostly disordered when bound to detergent micelles, in contrast to the highly α-helical structures found for most amyloid proteins. To determine the origin of this difference, the structures of PAP(248-286) were solved in aqueous solution and with 30% and 50% trifluoroethanol. In solution, pulsed field gradient (PFG)-NMR and (1)H-(1)H NOESY experiments indicate that PAP(248-286) is unfolded to an unusual degree for an amyloidogenic peptide but adopts significantly helical structures in TFE solutions. The clear differences between the structures of PAP(248-286) in TFE and SDS indicate electrostatic interactions play a large role in the folding of the peptide, consistent with the slight degree of penetration of PAP(248-286) into the hydrophobic core of the micelle. This is another noticeable difference between PAP(248-286) and other amyloid peptides, which generally show penetration into at least the headgroup region of the bilayer, and may explain some of the unusual properties of SEVI.

A proton spin diffusion based solid-state NMR approach for structural studies on aligned samples.

Rapidly expanding research on nonsoluble and noncrystalline chemical and biological materials necessitates sophisticated techniques to image these materials at atomic-level resolution. Although their study poses a formidable challenge, solid-state NMR is a powerful tool that has demonstrated application to the investigation of their molecular architecture and functioning. In particular, 2D separated-local-field (SLF) spectroscopy is increasingly applied to obtain high-resolution molecular images of these materials. However, despite the common use of SLF experiments in the structural studies of a variety of aligned molecules, the lack of a resonance assignment approach has been a major disadvantage. As a result, solid-state NMR studies have mostly been limited to aligned systems that are labeled with an isotope at a single site. Here, we demonstrate an approach for resonance assignment through a controlled reintroduction of proton spin diffusion in the 2D proton-evolved-local-field (PELF) pulse sequence. Experimental results and simulations suggest that the use of spin diffusion also enables the measurement of long-range heteronuclear dipolar couplings that can be used as additional constraints in the structural and dynamical studies of aligned molecules. The new method is used to determine the de novo atomic-level resolution structure of a liquid crystalline material, N-(4-methoxybenzylidene)-4-butylaniline, and its use on magnetically aligned bicelles is also demonstrated. We expect this technique to also be valuable in the structural studies of functional molecules like columnar liquid crystals and other biomaterials.