The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions

The amide I spectrum of parallel β-sheet proteins

The amide I absorption of the polypeptide backbone has long been used to analyze the secondary structure of proteins. This approach has gained additional attention in the context of amyloid diseases where a particular focus is on the distinction between parallel and antiparallel β-sheets because these structures often discriminate between pre-fibrillar structures and fibrils. Some earlier infrared spectra with typical features of antiparallel β-sheets were interpreted as arising from the parallel β-sheets of fibrils. Therefore, the ability of infrared spectroscopy to distinguish between both types of β-sheets is debated. While it is established that regular, antiparallel β-sheets give rise to a high wavenumber band near 1690 cm-1, it is less clear whether or not this band may also occur for parallel β-sheets. Here we present and analyze the amide I spectra of two β-helix proteins, SV2 and Pent. The overall shape of the proteins is that of a cuboid which has parallel β-sheets on its four sides, which are connected by bends. The main features of their amide I spectrum are a band at 1665, and two bands between 1645 and 1628 cm-1. Both proteins exhibit also a weak component band near 1690 cm-1. Calculations of the amide I spectrum indicate that the absorption at high wavenumbers is not caused by the parallel β-sheets but by the bends between the β-strands. We therefore suggest to modify the interpretation of the amide I spectrum as follows: a high wavenumber band near 1690 cm-1 may be caused by other structures than antiparallel β-sheets. However, when the spectrum consists of only two distinct bands, one near 1690 cm-1 and one near 1630 cm-1, then an assignment to antiparallel β-sheets is consistent with the literature

The amide I spectrum of parallel β-sheet proteins

The amide I absorption of the polypeptide backbone has long been used to analyze the secondary structure of proteins. This approach has gained additional attention in the context of amyloid diseases where a particular focus is on the distinction between parallel and antiparallel β-sheets because these structures often discriminate between pre-fibrillar structures and fibrils. Some earlier infrared spectra with typical features of antiparallel β-sheets were interpreted as arising from the parallel β-sheets of fibrils. Therefore, the ability of infrared spectroscopy to distinguish between both types of β-sheets is debated. While it is established that regular, antiparallel β-sheets give rise to a high wavenumber band near 1690 cm-1, it is less clear whether or not this band may also occur for parallel β-sheets. Here we present and analyze the amide I spectra of two β-helix proteins, SV2 and Pent. The overall shape of the proteins is that of a cuboid which has parallel β-sheets on its four sides, which are connected by bends. The main features of their amide I spectrum are a band at 1665, and two bands between 1645 and 1628 cm-1. Both proteins exhibit also a weak component band near 1690 cm-1. Calculations of the amide I spectrum indicate that the absorption at high wavenumbers is not caused by the parallel β-sheets but by the bends between the β-strands. We therefore suggest to modify the interpretation of the amide I spectrum as follows: a high wavenumber band near 1690 cm-1 may be caused by other structures than antiparallel β-sheets. However, when the spectrum consists of only two distinct bands, one near 1690 cm-1 and one near 1630 cm-1, then an assignment to antiparallel β-sheets is consistent with the literature

The amide I spectrum of parallel β-sheet proteins

The amide I absorption of the polypeptide backbone has long been used to analyze the secondary structure of proteins. This approach has gained additional attention in the context of amyloid diseases where a particular focus is on the distinction between parallel and antiparallel β-sheets because these structures often discriminate between pre-fibrillar structures and fibrils. Some earlier infrared spectra with typical features of antiparallel β-sheets were interpreted as arising from the parallel β-sheets of fibrils. Therefore, the ability of infrared spectroscopy to distinguish between both types of β-sheets is debated. While it is established that regular, antiparallel β-sheets give rise to a high wavenumber band near 1690 cm-1, it is less clear whether or not this band may also occur for parallel β-sheets. Here we present and analyze the amide I spectra of two β-helix proteins, SV2 and Pent. The overall shape of the proteins is that of a cuboid which has parallel β-sheets on its four sides, which are connected by bends. The main features of their amide I spectrum are a band at 1665, and two bands between 1645 and 1628 cm-1. Both proteins exhibit also a weak component band near 1690 cm-1. Calculations of the amide I spectrum indicate that the absorption at high wavenumbers is not caused by the parallel β-sheets but by the bends between the β-strands. We therefore suggest to modify the interpretation of the amide I spectrum as follows: a high wavenumber band near 1690 cm-1 may be caused by other structures than antiparallel β-sheets. However, when the spectrum consists of only two distinct bands, one near 1690 cm-1 and one near 1630 cm-1, then an assignment to antiparallel β-sheets is consistent with the literature