Elucidating the structure of an infectious protein

1 Deutsches Zentrum für Neurodegenerative Erkrankungen, Göttingen, Germany, 2 Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, 3 University Medical Center Göttingen, University of Göttingen, Göttingen, Germany, 4 CIMUS Biomedical Research Institute, University of Santiago de Compostela-IDIS, Santiago de Compostela, Spain, 5 Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, 6 Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada

Editor: Heather L. True, Washington University School of Medicine, UNITED STATES these cases, image processing takes advantage of the repeating structure and can extract molecular details through averaging that are not readily visible. Electron tomography can rapidly provide 3-D tomograms of the observed specimens, but the dose fractionation that is necessary to collect the different view angles limits the resolution of the reconstructed volumes [9,10].
High-resolution electron microscopy studies require the use of cryo low-dose imaging techniques. With the advent of direct electron detectors, unprecedented structural detail can be visualized, which, under optimal conditions, can reach atomic resolution [11,12]. The added sensitivity that is provided by these new detectors is revolutionizing electron cryomicroscopy and the structural details that can be obtained from even challenging samples, such as protein aggregates (Fig 1) [10]. However, the structural heterogeneity that is commonly seen with protein aggregates often limits the resolution that can be obtained, and this also applies to individual amyloid fibrils of infectious prions [10,13].

Diffraction techniques
Protein aggregates, including those composed of infectious proteins, are also amenable to structural analyses via diffraction techniques, such as small angle X-ray scattering (SAXS), Xray crystallography, and X-ray fiber diffraction. The latter technique is often used to detect the characteristic 4.8 Å cross-β signature of amyloid fibrils [14], which is a defining criterion commonly used in biophysics for the term "amyloid." The need to achieve sufficient sample orientation is an ongoing challenge for X-ray fiber diffraction analyses of amyloid fibrils, but welloriented samples can reveal the necessary structural details to define the molecular dimensions and structural architecture of different amyloid forms [15,16]. X-ray crystallography has more stringent demands, as it requires the protein to form well-ordered 3-D crystals, which is nearly impossible to achieve except with small amyloidogenic peptides [17]. In contrast, SAXS provides a measure of the sample/aggregate size without the need for sample orientation. In fact, the random orientation of the protein aggregates in solution allows calculation of the overall aspect ratio of the aggregate. Therefore, a sufficiently dispersed sample can provide molecular or protein aggregate dimensions via the radius of gyration [18].

Nuclear magnetic resonance spectroscopy
A large array of spectroscopic techniques has been applied to the study of protein aggregates, including infectious prions. One particularly powerful technique is nuclear magnetic resonance (NMR), because it provides single-residue resolution and can be applied to both soluble species and insoluble aggregates. In case of insoluble particles, the slow tumbling time of the aggregates has to be taken into account. Therefore, solution-state NMR is best combined with hydrogen/deuterium exchange assays, in which the exchange rate of hydrogen atoms, which participate in peptide bonds, with the solvent strongly depends on the local secondary structure in the aggregate. In particular, hydrogen atoms within β-strands participate in relatively stable hydrogen bonds and exchange very slowly. This makes hydrogen/deuterium exchange coupled with solution NMR spectroscopy a powerful tool to identify the location of the regular secondary structure elements in prion aggregates [19].
The most powerful experimental technique to characterize heterogeneous protein aggregates and infectious prions to date is solid-state NMR spectroscopy [20,21]. Solid-state NMR spectroscopy has now reached a level at which it can reliably determine the 3-D structure of single molecules in amyloid fibrils [22][23][24][25]. In combination with information from other techniques, such as electron microscopy and modeling, this approach can also provide insights into the higher order arrangements of molecules in prion aggregates [26,27].

Mass spectrometry
Mass spectrometry analysis of peptide fragments obtained under denaturing, exchangequenching conditions has been used to assess the global exchange of short stretches of a given protein. Application of such an approach to GPI-anchorless PrP Sc showed an overall very low rate of exchange of a stretch spanning from position~81 to~226, which is suggestive of a high content of β-sheet secondary structure and tight packing. Slightly higher exchange of some short regions within this stretch suggests the presence of short loops connecting short βstrands [28]. Similar results have been obtained more recently for infectious, recombinant PrP Sc , suggesting a common structure for all infectious PrP Sc forms [29], which is very different from that of noninfectious recombinant amyloids [28].

Chemical probes
Chemical probes have been successfully used to obtain structural information of proteins difficult to study by other means. Typical approaches include surface labeling, which provides information about accessibility of specific amino acids, and cross-linking with bifunctional reagents, which provides upper limits on the distance between pairs of accessible residues. Identification of modified sites is typically achieved by mass spectrometry after tryptic digestion [30].
Surface labeling of PrP Sc with tyrosine-specific reagents showed that its C-terminal region has suffered a very substantial structural rearrangement, contrary to the hypothesis of conserved C-terminal α-helices [31]. PrP Sc has also been probed with cross-linking reagents. Experiments using bis (sulfosuccinimidyl) suberate (BS 3 ) showed that the amino termini of successive PrP 27-30 units in a PrP 27-30 stack are within 11.4 Å [32]. While such a distance constraint was interpreted at the time as a limitation to the maximum number of rungs in the PrP Sc β-solenoid, it is fully compatible with head-to-head stacking of PrP Sc units [10].
In summary, chemical probing should be seen as a complement to other techniques that provide a general view of the architecture of infectious proteins. Recent advances in sensitivity and accuracy of mass spectrometry methods, such as Fourier-transform instruments and chemical footprinting with synchrotron radiation, has opened up exciting new possibilities [30].

Conclusion
Recent technological advances have provided a wealth of data on the structures of pathologically aggregated, infectious proteins involved in Alzheimer disease, Parkinson disease, and the prion diseases. In the former cases, the structure of the aggregated proteins (Aβ and αsynuclein) were found to adopt an in-register β-sheet structure [24,25]. In contrast, for the archetypical prion diseases (PrP Sc ), a four-rung β-solenoid architecture was observed [10,15], in agreement with lower-resolution approaches [18,28,31,32]. The ability to generate diseaserelevant protein conformers in vitro, in combination with solid-state NMR and other analysis techniques, was crucial for determining the high-resolution structures of misfolded Aβ and αsynuclein [24,25]. A similar approach may provide high-resolution structural information about PrP Sc in the future.