Infrared Spectroscopic Studies of Lyophilization‐ and Temperature‐Induced Protein Aggregation

A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.glucocorticoid receptor ͉ N-terminal activation function ͉ coregulatory protein ͉ protein folding T he glucocorticoid receptor (GR) is a ligand-activated transcription factor with the domain structural arrangement typical of the nuclear hormone receptors superfamily (1-4). The GR regulates transcription of target genes by binding DNA at specific hormone response elements and͞or by interacting with other transcription factors (5-8). Although the structural organization of steroid receptors into N-terminal domain (NTD), DNA binding domain (DBD), and ligand binding domain is well characterized (9-14), precisely how transcription is regulated by them is largely unknown. For all steroid receptors, this is in part due to the lack of information about their transcription activating domain AF1, located in the NTD (9, 10). The GR AF1 sequence resembles those of acidic transactivation domains of several transcription factors (15, 16). When expressed independently, the GR AF1 shows little structure and seems to exist as an ensemble of largely unstructured conformers (17, 18). The GR AF1 is known to interact with other transcription factors, and conditional folding has been suggested to be the key for these interactions (19)(20)(21)(22).It has been reported that in the presence of trifluoroethanol, the ''core'' of AF1 (AF1 c , amino acids 187-242), located toward the C-terminal end of AF1, adopts three helical segments (17). Independent experiments have shown that substitution of the ␣-helixbreaking amino acid proline for natural residues a...

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“…All second-derivative spectra were baseline-corrected and areanormalized as described in ref. 34. Good signal-to-noise ratios were obtained.…”

Section: Methodsmentioning

confidence: 86%

TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain

Kumar

Volk

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…We suggest that polypeptide fibrils can be placed into three structural groups: 1) protein fibrils, which consist of assemblies of globular protein units, such as actin or tubulin; 2) helical fibrils, for example collagen and intermediate filaments; and 3) fibrillar aggregates, such as amyloid fibrils, in which at least part of the native protein structure is lost. These amyloid fibrils are characterized by the presence of a specific type of intermolecular ␤-sheet conformation (21,22) and differ fundamentally from globular protein structures (28). The structures of globular proteins are determined by the interactions and packing of the side chains of the constituting amino acids and, hence, by the polypeptide sequence.…”

Section: Discussionmentioning

confidence: 99%

“…Consistent with this observation, we could not obtain evidence for the presence of a cross-␤ structure when we examined the fibril pellet with x-ray diffraction (data not shown), and also FTIR spectroscopy could not reveal aggregated ␤-sheet structure in QHF-litho. The sheets of amyloid or prion fibrils appear in the infrared spectrum with an amide I maximum close to 1620 cm Ϫ1 (11,21,22). In the case of QHFlitho, no such maximum could be discerned.…”

Section: Lithostathine Fibrils Did Not Possess the Classical Propertiesmentioning

confidence: 99%

Lithostathine Quadruple-helical Filaments Form Proteinase K-resistant Deposits in Creutzfeldt-Jakob Disease

Laurine

Grégoire

Fändrich³

et al. 2003

Journal of Biological Chemistry

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Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Strä ussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6 ]bibenzothiazolyl-2,2 -diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

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“…Changes in the distribution of secondary structures of freeze-dried proteins are most commonly assessed by visual comparison of the second derivative of the amide I band (1700-1600 cm -1 ) in the blank corrected FTIR spectra of the sample with a native reference spectrum [10,15,25,26,27,28,29,30]. Freeze-dried formulations were designated as NL if the amide I spectrum was similar (in secondary structural elements distribution) to that of the native LDH before freeze-drying (Fig.…”

Section: Ftir As a Reference Technique To Assign Class Labelsmentioning

confidence: 99%

Raman spectroscopy and multivariate analysis for the rapid discrimination between native-like and non-native states in freeze-dried protein formulations

Pieters

Heyden

Roger³

et al. 2013

European Journal of Pharmaceutics and Biopharmaceutics

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Infrared Spectroscopic Studies of Lyophilization‐ and Temperature‐Induced Protein Aggregation

Cited by 433 publications

References 52 publications

TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain

TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain

Lithostathine Quadruple-helical Filaments Form Proteinase K-resistant Deposits in Creutzfeldt-Jakob Disease

Raman spectroscopy and multivariate analysis for the rapid discrimination between native-like and non-native states in freeze-dried protein formulations

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