Comparison of LUT plots for dodecamers of SP-D seemed like a worthwhile test before going further on measuring the multimers and the trimers and the hexamers for peaks and valleys in grayscale.

So i picked on of the dodecamers that I though was clean and informative from a higher mag AFMicrograph and compared it at the same (as best as i could from the measurements on posted images (low res image coming from Hartshorn et al, 2008) to see whether the fuzzier and lower contrast image would produce the two main peaks plus three intermediate peaks in each arm of the dodecamer. (main central peak is combined from the Ntermini of two trimers or 4 trimers or many trimers in a fuzzyball – i don’t think it is certain yet whether the multimers are made of just hexamers or dodecamers or both).

But as you can see from LUT plots (using ImageJ) isolated straightened arms of these two dodecamers can be easily seen as pretty similar.  The one from the higher mag image is much nicer, but data are still present in the low mag image. So that is kind of nice since i can still use those to add to the dataset on positions and incidence of intermediate LUT peaks between the Ntermini and CRD.

Plots adjusted for half width are just under the copies of the two AFMicrographs…. best res on top, lowest res on bottom of that top image. and beneath that the adjusted best half LUT plot for each orange and purple respectively.  The first peak in the collagen-like domain is very very constant, also prominent. Staggered connections (as would be the Ntermini) are common in biology, especially the structural proteins.

LUT plots for SP-D trimers (n=9) all from same micrograph. There is some difference in my own perception of three top images on right hand said… something missing in the Nterminus region of the images. Finding out the width in nm of the left side (Ntermini) of these images can be done, but where to draw the line for the margin of each peak is a little bit subjective. (Image from Arroyo et al).
This image is the same as previous plot with the top two right plots added. Those LUT plots which show a “togetherness” of Nterminus and first part of collagen like portion are in blue while those plots in white have a distinct peak which corresponds to the first portion of the collagen-like domain.

From a different photo in this article there are trimers which have been exposed to pH (4.2) and heat (45^oC) which still to me have a similar LUT plot pattern (which I will do) to those not heat and pH stressed.  There are a couple however which do not show enough definition obtained with AFM (at least in their image (not posted here, but will post) image) in the CRD to distinguish it from the Nterminus.
Seems kind of interesting to speculate that the structure of hexamers of SP-D tend to make “v” shapes where there are two arms on either side of the point of the V…. which would indicate that dependent upon pH the fuzzyball might actually have side to side attachments of the Nterminus, thus corroborating the “empty” dark (low brightness or luminance) of the center of some fuzzball images. (low pH in some disease states)

Two peaks in the longer trimers at the Nterminus and first portion of the collagen like domain (trimers with white LUT plots over the exact image), but just one in the shorter trimers where it could be that the Nterminus and first portion of the collagen-like domain have become one peak. (blue LUT plot). Ntermini are on the left, CRD are on the right of each image. See the direction of the line segments used to determine the total length of the trimers that were curved on previous blog post.

Trimers of surfactant protein D gathered from a single image produced by Arroyo et al have been lined up and measured by discontinuous lines sums (to accommodate the curvature of some of the molecules). CRD are on the right hand sides of each of the 9 images, Ntermini on the left. It is clear that the 5-peak grayscale (LUT plot) motif is present on those images with a green spot but not really present on the images with the pink spot — where the Nterminus and the first part (peak) of the collagen-like domain appear as a “bent” or “scrunched” bright (in terms of grayscale) area and they are also significantly different (n=3 56.66nm+/- 3.23 vs n=6 71.82nm +/- 6.6, p=0.008). The selection looks biased (and is) but is based on the number of bright peaks perceived by naked eye. I will plot each and see reevaluate in next post. Also the shortest set have a greater brightness indicating greater height in AFM.

Another interesting thing is that the Ntermini in the trimers is NOT the brightest peak (which it is in all the multimers above that (hexamers, dodecamers, fuzzyballs) and this to me means that there is a side-to-side attachment of the Ntermini… but which looks different than the CRD’s obvious 3-lump images, it looks more like an overlapping splice joint  rather than half lap or side to side joint.

One question about SP-D fuzzyball multimers is whether or not the trimers, or hexamers or dodecamers only have N termini binding end-to-end making the diameter of the multimer about 120nm or whether it is possible that is there another configuration. SP-D multimers with fewer arms (maybe less than 12 or 14 often show a lucent (NOT MEANING luminous but the opposite, as in darker center… like electron lucent in TEM where there is a more background grayscale than a high level of “lightness” or “brightness”. This is what I have seen in multimers of SP-D of low arm count and I am trying to figure out a configuration of the multimer that satisfies this arrangement. In an image of a hexamer (Arryoy et al) there is one which really speaks to the possibility that N termini can bind side by side. Granted, Arroyo et al manipulated the pH of the preparation, but pH changes would occur in vivo in surfactant and other fluids as well, and might just be a trigger for fuzzyball formation that has arms that do NOT span the center N to end of Ntermini, but create a center hollow area with N to side of N binding.
Here is their picture…. N termini are the brightest portion on the lower part of the image. I did not ask for permission to repost this image but thank them.
and a cut and paste model from the above and a SP-D multimer from the same micrograph.  Aside from the butt and feet look