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Catalytic amyloid fibrils

Amyloid fibrils are formed from polypeptide chains assembled into an organized fibrillar structure. Now, it has been shown that such fibrillar structures can also bind metal ions and catalyse chemical reactions.

Tobias Aumüller and Marcus Fändrich

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in Nature Chemistry, Ivan V. Korendovych, William F. DeGrado and co-workers6 go one step beyond this by demonstrating an amyloid structural spine that converts into a catalytically active surface by binding of Zn2+ ions (Fig. 1c). The conceptual starting point of this development was the heptapeptide LKLKLKL, which was designed to possess an alternating pattern of hydrophobic and hydrophilic amino acid residues that promotes self-assembly into amyloidlike fibrils. This initial scaffold was then adapted to add catalytic activity. To achieve this, the team used a strategy inspired by a natural enzyme, archaeal γ-carbonic anhydrase, which is an efficient catalyst for the reversible hydration of carbon dioxide. The active site of this enzyme comprises a catalytically important Zn2+ ion that is bound by the imidazole groups from three His residues. By replacing the three Lys residues in their initial peptide with histidines and blocking the charged chemical groups at the N and C terminus, the team created a peptide with the sequence acetyl–LHLHLHL–amide. Circular dichroism experiments showed that this peptide is capable of forming aggregates containing β-sheets. Furthermore, in the presence of Zn2+, the

he self-assembly of polypeptide chains into linear amyloid fibrils is a fundamental biochemical transformation that is associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s1. These fibrils are defined by an extremely regular and highly periodic β-sheet conformation, which has led to suggestions that they could be used in a range of applications in material sciences and bionanotechnology 2,3. So far, fibrils have been adapted to form conductive nanowires, coating materials to promote the adhesion of cells in culture, nanostructured protein films, hydrogels and liquid crystal phases2,3. Amyloid-like fibrils have also been used as platforms for the immobilization of functional groups or catalytic protein domains. Previous efforts to functionalize fibrils have focused on the generation of fusion proteins between a globular protein of interest and an amyloid-forming peptide sequence, which serves as an anchor to incorporate the fusion protein within the structural spine of the fibril (Fig. 1a)4. Alternatively, functional groups or enzymatically active proteins can be added by crosslinking or chemical modification of the fully formed fibril or its structural precursors (Fig. 1b)5. Now, as they report a

b

CH2OH OH OH

CspB

dT7

+ O2

GOD

CspB

c OH

OH + H2O2

+

O

H2O

O

Zn2+

OH

NO2

Zn2+

CH2OH OH

dT7

O

aggregates could catalyse the hydrolysis of p-nitrophenyl acetate (pNPA) into nitrophenolate and acetate (Fig. 1c) — a reaction typically used to scrutinize the catalytic mechanisms of hydrolytic enzymes, including esterases and peptidases, as the reaction can easily be followed by absorption spectroscopy. Further evaluation of the catalytic properties of the acetyl– LHLHLHL–amide peptide according to Michaelis–Menten theory revealed a specificity constant (kcat/KM, where kcat is the turnover number and KM is the Michaelis constant) of 0.6 ± 0.08 M–1 s–1. To improve the catalytic activity, the authors systematically replaced the Leu or His residues with other amino acids, while retaining the overall pattern. The most active peptide identified by this strategy was acetyl–IHIHIQI–amide. Transmission electron microscopy confirmed that this peptide assembled into fibrils. Kinetic analysis, performed at variable substrate concentrations, verified the enzyme-like properties of the fibrils — such as observable saturation characteristics and a kcat/KM value of 62 ± 2 M–1 s–1 at pH 8.0 and of 360 ± 30 M–1 s–1 at pH 10.3. Although these values are lower than the value of kcat/KM reported for the hydrolysis of pNPA with human carbonic anhydrase C at pH 8.0 (~2,500 M–1 s–1) (ref. 7),

Zn2+

O O OH

Zn2+

HO

NO2 +

O OH

Figure 1 | Schematic representation of different modes of amyloid fibril functionalization. a, Display of cold shock protein B (CspB) on fibrils achieved by biotechnological fusion to a fibril-forming polypeptide (N-terminal domain of PABPN1 protein)4. Immobilized CspB retains affinity for the oligonucleotide 5’-TTTTTTT-3’ (dT7, green). b, Immobilization of glucose oxidase (GOD) on bovine insulin fibrils by glutaraldehyde crosslinking5. c, Korendovych, DeGrado and co-workers show that Zn2+-binding fibrils can hydrolyse pNPA into nitrophenole and acetic acid6. NATURE CHEMISTRY | VOL 6 | APRIL 2014 | www.nature.com/naturechemistry

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news & views the large number of catalytically active sites per fibril makes the catalytic performance of these structures, relative to their total protein mass, comparable to that of the natural carbonic anhydrase. The strategy by which Korendovych and co-workers engineered a self-assembling biocatalyst is astonishingly simple, because it relies on small peptide units that can be readily synthesized and modified by chemical methods. Peptide synthesis is not limited to proteinogenic amino acids, suggesting possibilities to use this methodology to generate chemical functionalities that are absent in natural proteins. As impressive as this work is, there are still some considerable challenges remaining: (i) to prove that the design principle can be used to engineer catalysts for other — and perhaps more complex — chemical reactions; (ii) to increase the catalytic efficiency; and (iii) to rationally design specificity for certain substrate molecules. The latter property has obviously been a crucial factor in the evolution of biological enzymes. In an enzyme’s active

site, specificity depends on the formation of a complex network of chemical interactions between the enzyme and substrate, often with one or both partners adopting a favourable conformation (known as an induced fit). Yet amyloid fibrils are comparatively rigid structures that possess rather smooth surfaces at their spines and lack deep cavities. This rigidity may be an additional burden when attempting to improve on catalytic efficiency, if the latter requires dynamically folded protein structures8. Finally, it is tempting to speculate as to whether amyloid toxicity in human diseases arises, at least in part, from the ability of amyloid structures to bind to metal ions that then allow new, highly detrimental, catalytic activities. A related mechanism has previously been suggested for Aβ peptide, the amyloid-forming peptide in Alzheimer’s disease, which was shown to bind Zn2+, Cu2+ or other metal ions by a histidine-rich motif and to exhibit catalytic activity. In that example, however, the fibril promotes redox reactions and the formation of

harmful reactive oxygen species9. A better demonstration of the full pathophysiological relevance of these catalytic processes, within the context of the numerous toxicity mechanisms that have been suggested for amyloidogenic peptides, will remain an important goal of future research. ❐ Tobias Aumüller and Marcus Fändrich are at the Institute for Pharmaceutical Biotechnology, Ulm University, Helmholtzstrasse 8/1, 89081 Ulm, Germany. e-mail: [email protected]; [email protected] References 1. Chiti, F. & Dobson, C. M. Annu. Rev. Biochem. 75, 333–366 (2006). 2. Knowles, T. P. J. & Buehler, M. J. Nature Nanotech. 6, 469–479 (2011). 3. Cherny, I. & Gazit, E. Angew. Chem. Int. Ed. 47, 4062–4069 (2008). 4. Sackewitz, M. et al. Protein Sci. 17, 1044–1054 (2008). 5. Pilkington, S. M., Roberts, S. J., Meade, S. J. & Gerrard, J. A. Biotechnol. Prog. 26, 93–100 (2010). 6. Rufo, C. R. et al. Nature Chem. 6, 303–309 (2014). 7. Verpoorte, J. A., Mehta, S. & Edsall, J. T. J. Biol. Chem. 242, 4221–4229 (1967). 8. Eisenmesser, E. Z. et al. Nature 438, 117–121 (2005). 9. Cassagnes, L-E. et al. Angew. Chem. Int. Ed. 52, 11110–11113 (2013).

REACTION KINETICS

Isotope effects feel the cold

Kinetic isotope effects are widely used to elucidate reaction mechanisms and are generally interpreted in terms of simple kinetic models. Measurements of this effect for the Penning ionization reaction between helium and dihydrogen highlight the need for a quantum description of chemical reaction rates when sub-kelvin temperatures are approached.

Mark Brouard

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hen interpreting kinetic isotope effects for gas-phase chemical reactions one often resorts to simple models of reaction rates, such as transition state theory 1. These approximate theories are really only appropriate for reactions studied under relatively hightemperature conditions, at which quantum effects such as tunnelling or resonances are less important. Figure 1a illustrates the origin of the kinetic isotope effect for a direct reaction possessing a potential energy barrier of height ΔE. On the basis of transition state theory 1, the rate of a chemical reaction can be written as being proportional to e–ΔE/RT. Changes in barrier height therefore lead to exponential changes in the rate of chemical reaction. Within this model of thermal rate coefficients other factors may also play a role, such as the vibrational and rotational energy level structure, which would be accounted for by the partition functions 274

of the reactants and transition state. But differences in barrier heights associated with the zero-point vibrational energies of the species involved usually dominate the kinetic isotope effect. Within this simple treatment, reactions involving D2 as a reactant are typically slower than reactions involving H2, because D2 reactions tend to have lower zero-point energies and thus higher reaction barriers (Fig. 1a). However, this simple theoretical picture provides only an approximate description of chemical reaction rates and it is put under severe scrutiny when applied to reactions at very low temperatures. It is well known, for example, that as the temperature is lowered, quantum mechanical effects, such as tunnelling, play a more important role, and can lead to reaction rates that differ by many orders of magnitude from the values expected on the basis of the simple ideas outlined above. Now, writing in Nature Chemistry, Narevicius and colleagues

describe such unanticipated reaction rates for the Penning ionization process He* + H2 → He + H2+ + e– at milli-kelvin temperatures. Interestingly, they also see a kinetic isotope effect; however, its origins differ greatly from the standard kinetic isotope effect described above. To understand the phenomenon observed by Narevicius and colleagues, one must first understand what happens when reactants collide. Figure 1b illustrates the classical encounter between two reactants A and B, represented as structureless spheres2. The particles approach with a well-defined impact parameter, b, which determines whether the collision is head-on or glancing in nature. More formally, the impact parameter defines the magnitude of classical orbital angular momentum, |ℓ|, associated with the collision |ℓ| = μvb, where μ is the reduced mass of the reactant pair and v is their relative velocity. As the temperature of the

NATURE CHEMISTRY | VOL 6 | APRIL 2014 | www.nature.com/naturechemistry

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Protein chemistry: catalytic amyloid fibrils.

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