Dynamic light scattering and atomic force microscopy imaging on fragments of $\beta$-connectin from human cardiac muscle

In order to investigate the protein folding-unfolding process, dynamic light scattering (DLS) and atomic force microscopy (AFM) imaging were used to study two fragments of the muscle cardiac protein $\beta$-connectin, also known as titin. Both fragments belong to the $I$ band of the sarcomer, and they are composed of four domains from $I_{27}$ to $I_{30}$ (tetramer) and eight domains from $I_{27}$ to $I_{34}$ (octamer). DLS measurements provide the size of both fragments as a function of temperature from 20 up to 86 °C, and show a thermal denaturation due to temperature increase. AFM imaging of both fragments in the native state reveals a homogeneous and uniform distribution of comparable structures. The DLS and AFM techniques turn out to be complementary for size measurements of the fragments and fragment aggregates. An unexpected result is that the octamer folds into a smaller structure than the tetramer and the unfolded octamer is also smaller than the unfolded tetramer. This feature seems related to the significance of the hydrophobic interactions between domains of the fragment. The longer the fragment, the more easily the hydrophobic parts of the domains interact with each other. The fragment aggregation behavior, in particular conditions, is also revealed by both DLS and AFM as a process that is parallel to the folding-unfolding transition.

Figure 1

DLS normalized experimental correlation functions at 90° for tetramer solutions.

Figure 2

DLS normalized experimental correlation functions at 90° for octamer solutions.

Figure 3

Particle size distribution of tetramer solution at 20 °C at 90°. In Figs. 3, 4, 5, 6, 7, 8, the histogram maximum height is normalized to 100% by intensity.

Figure 4

Particle size distribution of tetramer solution at 86 °C at 90°.

Figure 5

Particle size distribution of octamer solutions at 20 °C at 90°.

Figure 6

Particle size distribution of octamer solutions at 86 °C at 90°.

Figure 7

Two populations, tetramers and aggregates, are shown at 20 °C and 20°. In the inset the fragment population is reported.

Figure 8

Two populations, octamers and aggregates, are shown at 20 °C and 20°. In the inset the fragment population is reported.

Figure 9

Translational diffusion coefficient of the fragment population in the whole angular range at 20 °C.

Figure 10

Translational diffusion coefficient of the fragment population at 20 °C, after thermal denaturation.

Figure 11

(Color online) AFM images of (a) tetramer and (b) octamer fragments deposited on mica substrate. Image size is $500\times 500{\text{nm}}^{\text{2}}$. $Z$ (vertical) ranges are 4.0 (a) and 4.1 (b) nm.

Figure 12

Histograms of length distribution for tetramers. For each histogram of Figs. 12, 13, 14, 15, 17, 18, 100 structures have been analyzed. The numerical counts represent how many structures with the size reported on the abscissa have been counted.

Figure 13

Histograms of width distribution for tetramers.

Figure 14

Histograms of length distribution for octamers.

Figure 15

Histograms of width distribution for octamers.

Figure 16

(Color online) (a) AFM image of octamer fragments near isoelectric point. Image size is $1.8\times 1.8\mu {\text{m}}^{\text{2}}$. $Z$ range is 23.7 nm. Two isolated aggregates of octamers are observable. (b) Three-dimensional zoom of image (a) on the smaller aggregate. (c), (d) Line profiles along big (c) and small (d) aggregates showing the protein height.

Figure 17

Histograms of length distribution for octamers near isoelectric point.

Figure 18

Histograms of width distribution for octamers near isoelectric point.