Insulin Assembly Damps Conformational Fluctuations: Raman Analysis of Amide I Linewidths in Native States and Fibrils

Dong, Jian; Wan, Zi Yi; Popov, Maxim N.; Carey, Paul R.; Weiss, Michael A.

doi:10.1016/s0022-2836(03)00536-9

Cited by 92 publications

(116 citation statements)

References 56 publications

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“…As observed in studies of insulin fibrils (15)(16)(17), Raman studies demonstrate that proinsulin can undergo a large scale conformational transition from ␣-helix to ␤-sheet. Whereas in native proinsulin the connecting peptide is largely disordered, analysis of amide Raman bands suggests that in fibrils the connecting peptide in part participates in a ␤-sheet.…”

mentioning

confidence: 71%

“…PBS provides a model for fibrillation of insulin in pharmaceutical formulations; the present solutions are made zinc-free to accelerate fibrillation. Acidic conditions are classic (6,7,35,36) and widely employed in structural studies (12,16,17). Fibrillation of insulin is more rapid under acidic conditions, presumably due to the absence of native self-association (37) and destabilization of the monomeric fold (38).…”

Section: Overviewmentioning

confidence: 99%

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Proinsulin Is Refractory to Protein Fibrillation

Huang

Dong

Phillips

et al. 2005

Journal of Biological Chemistry

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Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-␤ assembly. Protection from fibrillation in ␤ cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific ␤-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-␤ assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect ␤ cells from toxic protein misfolding in the endoplasmic reticulum.

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confidence: 71%

Section: Overviewmentioning

confidence: 99%

Proinsulin Is Refractory to Protein Fibrillation

Huang

Dong

Phillips

et al. 2005

Journal of Biological Chemistry

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“…Raman spectroscopy enabled elucidation of the fibrillation mechanism and revealed chemical/structural information that would have been difficult to obtain with any other technique. 130,189 In 2013, the Goodacre group used 2D correlation analysis of CRS data (780 nm excitation) to probe the stability of ribonuclease proteins. They showed it was possible to compare the unfolding profiles of the proteins and to show very subtle protein conformational changes that could not observed with a spectrofluorophotometer.…”

Section: Analysis Of Biopharmaceutical Productsmentioning

confidence: 99%

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

2017

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The production of active pharmaceutical ingredients (APIs) is currently undergoing its biggest transformation in a century. The changes are based on the rapid and dramatic introduction of protein-and macromolecule-based drugs (collectively known as biopharmaceuticals) and can be traced back to the huge investment in biomedical science (in particular in genomics and proteomics) that has been ongoing since the 1970s. Biopharmaceuticals (or biologics) are manufactured using biological-expression systems (such as mammalian, bacterial, insect cells, etc.) and have spawned a large (>E35 billion sales annually in Europe) and growing biopharmaceutical industry (BioPharma). The structural and chemical complexity of biologics, combined with the intricacy of cell-based manufacturing, imposes a huge analytical burden to correctly characterize and quantify both processes (upstream) and products (downstream). In small molecule manufacturing, advances in analytical and computational methods have been extensively exploited to generate process analytical technologies (PAT) that are now used for routine process control, leading to more efficient processes and safer medicines. In the analytical domain, biologic manufacturing is considerably behind and there is both a huge scope and need to produce relevant PAT tools with which to better control processes, and better characterize product macromolecules. Raman spectroscopy, a vibrational spectroscopy with a number of useful properties (nondestructive, non-contact, robustness) has significant potential advantages in BioPharma. Key among them are intrinsically high molecular specificity, the ability to measure in water, the requirement for minimal (or no) sample pre-treatment, the flexibility of sampling configurations, and suitability for automation. Here, we review and discuss a representative selection of the more important Raman applications in BioPharma (with particular emphasis on mammalian cell culture). The review shows that the properties of Raman have been successfully exploited to deliver unique and useful analytical solutions, particularly for online process monitoring. However, it also shows that its inherent susceptibility to fluorescence interference and the weakness of the Raman effect mean that it can never be a panacea. In particular, Raman-based methods are intrinsically limited by the chemical complexity and wide analyte-concentration-profiles of cell culture media/bioprocessing broths which limit their use for quantitative analysis. Nevertheless, with appropriate foreknowledge of these limitations and good experimental design, robust analytical methods can be produced. In addition, new technological developments such as time-resolved detectors, advanced lasers, and plasmonics offer potential of new Raman-based methods to resolve existing limitations and/or provide new analytical insights.

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“…Nearby silk units will bind to the template to form a critical size for nucleation. Inter-or intramolecular interactions between multiple SELPs stretched together or local fluctuations of conformational states cause growth of assembled nuclei through an induced fit mechanism using a template formed during stretching [76].…”

Section: Speculation On Mechanisms Of Nucleation Formation Under Nanomentioning

confidence: 99%

Direct Observation of Amyloid Nucleation under Nanomechanical Stretching

et al. 2013

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Self-assembly of amyloid nanofiber is associated with functional and pathological processes such as in neurodegenerative diseases. Despite intensive studies, stochastic nature of the process has made it difficult to elucidate molecular mechanisms for the key amyloid nucleation. Here, we investigated the amyloid nucleation of silk-elastinlike peptide (SELP) using time-lapse lateral force microscopy (LFM). By repeated scanning a single line on a SELP-coated mica surface, we observed sudden stepwise height increases, corresponds to nucleation of an amyloid fiber. The lateral force profiles followed either a worm-like chain model or an exponential function, suggesting that the atomic force microscopy (AFM) tip stretches a single or multiple SELP molecules along the scanning direction, serves as the template for further selfassembly perpendicular to the scan direction. Such mechanically induced nucleation of amyloid fibrils allows positional and directional control of amyloid assembly in vitro, which we demonstrate by generating single nanofibers at predetermined nucleation sites.

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Insulin Assembly Damps Conformational Fluctuations: Raman Analysis of Amide I Linewidths in Native States and Fibrils

Cited by 92 publications

References 56 publications

Proinsulin Is Refractory to Protein Fibrillation

Proinsulin Is Refractory to Protein Fibrillation

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

Direct Observation of Amyloid Nucleation under Nanomechanical Stretching

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