Ralf Langen

Recent advances in site-directed spin labeling of proteins

Site-directed spin labeling of proteins is experiencing a phase of rapid technical evolution, application and evaluation. New strategies have been introduced for determining membrane protein topography, electrostatic potentials, the orientation of proteins at membrane surfaces and inter-residue distances. New applications include studies of beta strands, structure mapping using spin-spin interactions, domain motions in soluble proteins and extensive structural analysis of a number of membrane and soluble proteins.

Mechanism of endophilin N-BAR domain-mediated membrane curvature.

Endophilin‐A1 is a BAR domain‐containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C‐terminal SH3 domain. We examine the mechanism by which the BAR domain and an N‐terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N‐BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin‐A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N‐BAR domains function as low‐affinity dimers regulating binding partner recruitment to areas of high membrane curvature.

Structural and dynamic features of Alzheimer's Aβ peptide in amyloid fibrils studied by site-directed spin labeling

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimers amyloid β (Aβ) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23–26). Despite their different aggregation properties and roles in disease, the two peptides, Aβ40 and Aβ42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.

Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement

α-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, α-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of α-synuclein bound to a lipid bilayer. Continuous-wave EPR was used to assign local secondary structure and to determine the membrane immersion depth of lipid-exposed residues, whereas pulsed EPR was used to map long-range distances. The structure of α-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that α-synuclein forms an extended, curved α-helical structure that is over 90 aa in length. The monomeric helix has a superhelical twist similar to that of right-handed coiled-coils which, like α-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd = 0.82 Å). The α-synuclein helix extends parallel to the curved membrane in a manner that allows conserved Lys and Glu residues to interact with the zwitterionic headgroups, while uncharged residues penetrate into the acyl chain region. This structural arrangement is significantly different from that of α-synuclein in the presence of the commonly used membrane-mimetic detergent SDS, which induces the formation of two antiparallel helices. Our structural analysis emphasizes the importance of studying membrane protein structure in a bilayer environment.

Structural Organization of α-Synuclein Fibrils Studied by Site-directed Spin Labeling

Despite its importance in Parkinsons disease, a detailed understanding of the structure and mechanism of α-synuclein fibril formation remains elusive. In this study, we used site-directed spin labeling and electron paramagnetic resonance spectroscopy to study the structural features of monomeric and fibrillar α-synuclein. Our results indicate that monomeric α-synuclein, in solution, has a highly dynamic structure, in agreement with the notion that α-synuclein is a natively unfolded protein. In contrast, fibrillar aggregates of α-synuclein exhibit a distinct domain organization. Our data identify a highly ordered and specifically folded central core region of ∼70 amino acids, whereas the N terminus is structurally more heterogeneous and the C terminus (∼40 amino acids) is completely unfolded. Interestingly, the central core region of α-synuclein exhibits several features reminiscent of those observed in the core region of fibrillar Alzheimers amyloid β peptide, including an in-register parallel structure. Although the lengths of the respective core regions differ, fibrils from different amyloid proteins nevertheless appear to be able to take up highly similar, and possibly conserved, structures.

Investigation of α-Synuclein Fibril Structure by Site-directed Spin Labeling

The misfolding and fibril formation of α-synuclein plays an important role in neurodegenerative diseases such as Parkinson disease. Here we used electron paramagnetic resonance spectroscopy, together with site-directed spin labeling, to investigate the structural features of α-synuclein fibrils. We generated fibrils from a total of 83 different spin-labeled derivatives and observed single-line, exchange-narrowed EPR spectra for the majority of all sites located within the core region of α-synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of α-synuclein fibrils must therefore be arranged in a parallel, in-register structure wherein same residues from different molecules are stacked on top of each other. This parallel, in-register core region extends from residue 36 to residue 98 and is tightly packed. Only a few sites within the core region, such as residues 62–67 located at the beginning of the NAC region, as well as the N- and C-terminal regions outside the core region, are significantly less ordered. Together with the accessibility measurements that suggest the location of potential β-sheet regions within the fibril, the data provide significant structural constraints for generating three-dimensional models. Furthermore, the data support the emerging view that parallel, in-register structure is a common feature shared by a number of naturally occurring amyloid fibrils.

Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.

Drusen deposits associated with aging and age-related macular degeneration contain nonfibrillar amyloid oligomers

Protein misfolding and aggregation are thought to underlie the pathogenesis of many amyloid diseases, such as Alzheimer and Parkinson diseases, whereby a stepwise protein misfolding process begins with the conversion of soluble protein monomers to prefibrillar oligomers and progresses to the formation of insoluble amyloid fibrils. Drusen are extracellular deposits found in aging eyes and in eyes afflicted with age-related macular degeneration (AMD). Recent characterizations of drusen have revealed protein components that are shared with amyloid deposits. However, characteristic amyloid fibrils have thus far not been identified in drusen. In this study, we tested the hypothesis that nonfibrillar oligomers may be a common link in amyloid diseases. Oligomers consisting of distinct amyloidogenic proteins and peptides can be detected by a recently developed antibody that is thought to recognize a common structure. Notably, oligomers exhibit cellular toxicity, which suggests that they play a role in the pathogenesis of neurodegenerative diseases. Through use of the anti-oligomer antibody, we came to observe the presence of nonfibrillar, toxic oligomers in drusen. Conversely, no reactivity was observed in age-matched control eyes without drusen. These results suggest that amyloid oligomers may be involved in drusen biogenesis and that similar protein misfolding processes may occur in AMD and amyloid diseases.

Structure of α-Helical Membrane-bound Human Islet Amyloid Polypeptide and Its Implications for Membrane-mediated Misfolding

Human islet amyloid polypeptide (hIAPP) misfolding is thought to play an important role in the pathogenesis of type II diabetes mellitus. It has recently been shown that membranes can catalyze the misfolding of hIAPP via an α-helical intermediate of unknown structure. To better understand the mechanism of membrane-mediated misfolding, we used site-directed spin labeling and EPR spectroscopy to generate a three-dimensional structural model of this membrane-bound form. We find that hIAPP forms a single α-helix encompassing residues 9–22. The helix is flanked by N- and C-terminal regions that do not take up a clearly detectable secondary structure and are less ordered. Residues 21 and 22 are located in a transitional region between the α-helical structure and C terminus and exhibit significant mobility. The α-helical structure presented here has important implications for membrane-mediated aggregation. Anchoring hIAPP to the membrane not only increases the local concentration but also reduces the encounter between peptides to essentially a two-dimensional process. It is significant to note that the α-helical membrane-bound form leaves much of an important amyloidogenic region of hIAPP (residues 20–29) exposed for misfolding. Misfolding of this and other regions is likely further aided by the low dielectric environment near the membrane that is known to promote secondary structure formation. Based upon these considerations, a structural model for membrane-mediated aggregation is discussed.

Fibrils with parallel in-register structure constitute a major class of amyloid fibrils: molecular insights from electron paramagnetic resonance spectroscopy.

The deposition of amyloid- and amyloid-like fibrils is the main pathological hallmark of numerous protein misfolding diseases including Alzheimers disease, transmissible spongiform encephalopathy, and type 2 diabetes. Besides the well-established role in disease, recent work on a variety of organisms ranging from bacteria to humans suggests that amyloid fibrils can also convey biological functions. To better understand the molecular mechanisms by which amyloidogenic proteins misfold in disease or perform biological functions, structural information is essential. Although high-resolution structural analysis of amyloid fibrils has been challenging, a combination of biophysical approaches is beginning to unravel the various structural features of amyloid fibrils. Here we review these recent developments with particular emphasis on amyloid fibrils that have been studied using site-directed spin labeling and electron paramagnetic resonance spectroscopy. This approach has been used to define the precise location of fibril-forming core regions and identify local secondary structures within such core regions. Perhaps one of the most remarkable findings arrived at by site-directed spin labeling was that most fibrils that contain an extensive core region of 20 amino acids or more share a common parallel in-register arrangement of beta strands. The preference for this arrangement can be explained on topological grounds and may be rationalized by the maximization of hydrophobic contact surface.