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A monomer-trimer model supports intermittent glucagon fibril growth.

Košmrlj A, Cordsen P, Kyrsting A, Otzen DE, Oddershede LB, Jensen MH - Sci Rep (2015)

Bottom Line: The growth probability is large at high and low concentrations and is reduced for intermediate glucagon concentrations.To explain this behavior we propose a simple model, where fibrils come in two forms, one built entirely from glucagon monomers and one entirely from glucagon trimers.The opposite building blocks act as fibril growth blockers, and this generic model reproduces experimental behavior well.

View Article: PubMed Central - PubMed

Affiliation: Harvard University, Department of Physics, 17 Oxford Street, Cambridge, MA 02138, USA.

ABSTRACT
We investigate in vitro fibrillation kinetics of the hormone peptide glucagon at various concentrations using confocal microscopy and determine the glucagon fibril persistence length 60μm. At all concentrations we observe that periods of individual fibril growth are interrupted by periods of stasis. The growth probability is large at high and low concentrations and is reduced for intermediate glucagon concentrations. To explain this behavior we propose a simple model, where fibrils come in two forms, one built entirely from glucagon monomers and one entirely from glucagon trimers. The opposite building blocks act as fibril growth blockers, and this generic model reproduces experimental behavior well.

No MeSH data available.


Related in: MedlinePlus

(a–c): Confocal microscopy images of glucagon fibrils with initial concentration 3 mg/mL in aqueous buffer (50 mM glycine HCl, pH 2.5) at three consecutive times: 64, 87 and 126 mins after the onset of fibrillation. Scale bar shows 5 μm. Each circle represents a data point and the red line represents the cumulated tracked positions of the growing fibril end. (d) Growth of 20 fibrils at the glucagon concentration of 3 mg/mL. Plateaus correspond to arrested states while fibrils elongate outside the plateaus. (e) The average end-to-end-distance squared () as a function of fibril length. The solid green line is obtained by fitting Eq. (1) to combined experimental data (red points) from all glucagon concentrations. The persistence length of fibrils is returned by the fit as 60 ± 2μm.
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f1: (a–c): Confocal microscopy images of glucagon fibrils with initial concentration 3 mg/mL in aqueous buffer (50 mM glycine HCl, pH 2.5) at three consecutive times: 64, 87 and 126 mins after the onset of fibrillation. Scale bar shows 5 μm. Each circle represents a data point and the red line represents the cumulated tracked positions of the growing fibril end. (d) Growth of 20 fibrils at the glucagon concentration of 3 mg/mL. Plateaus correspond to arrested states while fibrils elongate outside the plateaus. (e) The average end-to-end-distance squared () as a function of fibril length. The solid green line is obtained by fitting Eq. (1) to combined experimental data (red points) from all glucagon concentrations. The persistence length of fibrils is returned by the fit as 60 ± 2μm.

Mentions: Here we significantly expand our previous work16 by monitoring fibril kinetics over a wide range of glucagon concentrations and by proposing a new model that captures the underlying molecular mechanisms of the process. This allows us to sample conditions spanning different precursor states of glucagon, i.e. monomers or trimers, leading to twisted or non-twisted fibrils, respectively. Fibrils were labeled with the fluorescent dye ThT and monitored using a confocal microscope with an Argon laser. On freshly plasmated glass plates we observed a volume of ~ 40 × 40 × 0.5 μm3. For each of the five different initial glucagon concentrations (1.5, 3, 6, 10 and 15 mg/mL), a minimum of two experiments were conducted in aqueous buffer (50 mM glycine HCl, pH 2.5). The time interval between captured frames was 3.3 mins and the total observation time of each experiment was about three days. When fibrils grew along the surface we tracked their length as a function of time. Sample images of real time growth of an individual fibril are shown in Fig. 1(a–c). The observed growing fibrils are relatively straight and their persistence length ℓp can be extracted by comparing the geometric distance between fibril ends Ree to the fibril length L. For semi-flexible fibrils the average end-to-end distance is expected to be17


A monomer-trimer model supports intermittent glucagon fibril growth.

Košmrlj A, Cordsen P, Kyrsting A, Otzen DE, Oddershede LB, Jensen MH - Sci Rep (2015)

(a–c): Confocal microscopy images of glucagon fibrils with initial concentration 3 mg/mL in aqueous buffer (50 mM glycine HCl, pH 2.5) at three consecutive times: 64, 87 and 126 mins after the onset of fibrillation. Scale bar shows 5 μm. Each circle represents a data point and the red line represents the cumulated tracked positions of the growing fibril end. (d) Growth of 20 fibrils at the glucagon concentration of 3 mg/mL. Plateaus correspond to arrested states while fibrils elongate outside the plateaus. (e) The average end-to-end-distance squared () as a function of fibril length. The solid green line is obtained by fitting Eq. (1) to combined experimental data (red points) from all glucagon concentrations. The persistence length of fibrils is returned by the fit as 60 ± 2μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355668&req=5

f1: (a–c): Confocal microscopy images of glucagon fibrils with initial concentration 3 mg/mL in aqueous buffer (50 mM glycine HCl, pH 2.5) at three consecutive times: 64, 87 and 126 mins after the onset of fibrillation. Scale bar shows 5 μm. Each circle represents a data point and the red line represents the cumulated tracked positions of the growing fibril end. (d) Growth of 20 fibrils at the glucagon concentration of 3 mg/mL. Plateaus correspond to arrested states while fibrils elongate outside the plateaus. (e) The average end-to-end-distance squared () as a function of fibril length. The solid green line is obtained by fitting Eq. (1) to combined experimental data (red points) from all glucagon concentrations. The persistence length of fibrils is returned by the fit as 60 ± 2μm.
Mentions: Here we significantly expand our previous work16 by monitoring fibril kinetics over a wide range of glucagon concentrations and by proposing a new model that captures the underlying molecular mechanisms of the process. This allows us to sample conditions spanning different precursor states of glucagon, i.e. monomers or trimers, leading to twisted or non-twisted fibrils, respectively. Fibrils were labeled with the fluorescent dye ThT and monitored using a confocal microscope with an Argon laser. On freshly plasmated glass plates we observed a volume of ~ 40 × 40 × 0.5 μm3. For each of the five different initial glucagon concentrations (1.5, 3, 6, 10 and 15 mg/mL), a minimum of two experiments were conducted in aqueous buffer (50 mM glycine HCl, pH 2.5). The time interval between captured frames was 3.3 mins and the total observation time of each experiment was about three days. When fibrils grew along the surface we tracked their length as a function of time. Sample images of real time growth of an individual fibril are shown in Fig. 1(a–c). The observed growing fibrils are relatively straight and their persistence length ℓp can be extracted by comparing the geometric distance between fibril ends Ree to the fibril length L. For semi-flexible fibrils the average end-to-end distance is expected to be17

Bottom Line: The growth probability is large at high and low concentrations and is reduced for intermediate glucagon concentrations.To explain this behavior we propose a simple model, where fibrils come in two forms, one built entirely from glucagon monomers and one entirely from glucagon trimers.The opposite building blocks act as fibril growth blockers, and this generic model reproduces experimental behavior well.

View Article: PubMed Central - PubMed

Affiliation: Harvard University, Department of Physics, 17 Oxford Street, Cambridge, MA 02138, USA.

ABSTRACT
We investigate in vitro fibrillation kinetics of the hormone peptide glucagon at various concentrations using confocal microscopy and determine the glucagon fibril persistence length 60μm. At all concentrations we observe that periods of individual fibril growth are interrupted by periods of stasis. The growth probability is large at high and low concentrations and is reduced for intermediate glucagon concentrations. To explain this behavior we propose a simple model, where fibrils come in two forms, one built entirely from glucagon monomers and one entirely from glucagon trimers. The opposite building blocks act as fibril growth blockers, and this generic model reproduces experimental behavior well.

No MeSH data available.


Related in: MedlinePlus