However, by the time I was called for an interview, I had decided to study protein structure and Caltech did not seem (based on the literature sent to me by the Biology Department) like a very good place to do that . There were several things I liked about the institute and the graduate program in the Division of Biology. I would like to thank Pamela Bjorkman, my dissertation advisor, for providing an environment conducive to learning and for being supportive and patient.
Patience is definitely a virtue, and while she often doesn't seem to be very patient, she is almost infinitely so when it really matters. I would like to thank the former and current (famous and notorious) members of the Bjorkman lab for providing interesting discussions and for helping me cope with day-to-day life in the lab. I thank my father, Bill, and my stepmother, Diana, for providing me with love, a great neighborhood shelter, companionship, good food, and a washing machine.
Finally, I would like to thank my wife, Wai-Sze Rachel Huber, who put up with my long work hours and experienced the frustration and brief moments of substitute. In photomicrographs, rotating shadow Ng and NgFnl-5 appear to be highly flexible rod-like molecules.
INTRODUCTION
Several members of the lgSF have been found to play important roles in the development of the nervous system. At least 27 alternatively spliced forms of the neural cell adhesion molecule mRNA are expressed during rat heart development. Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule.
Binding of the integrin Mac-1 (CD 11 b/CD 18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. The arrangement of the immunoglobulin-like domains of iCAM-1 and the binding sites for LFA-1 and rhinovirus. Identification of a heparin-binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides.
The 180-kD component of the neural cell adhesion molecule N-CAM is involved in intercellular contacts and cytoskeleton-membrane interactions. Different extracellular domains of neural cell adhesion molecule (N-CAM) are involved in different functions. The neural adhesion molecule L1 as a member of the immunoglobulin superfamily with fibronectin-like binding domains.
Drosophila Neuroglian: A member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule Ll.
CRYSTAL STRUCTURE OFT AND EM TYPE III FIDRONECTIN
SDS-polyacrylam1de gel electrophoresis (15%) of glycosylated and deglycosylated forms of the chymotrypt1c fragment of the five Fn-111 repeat proteins. A segment consisting of five Fn-111 repeats was then crystallized, but again these crystals did not diffract to atomic resolution. Sequence analysis revealed that the chymotryptic fragment begins at residue 610 and the tryptic fragment begins at 607, near the predicted start of the first repeat of Fn-111 (Bieber et al., 1989).
Neuroglial motifs of fn-111 have similar structures The structure of the proteolytic fragment of neuroglial repeats of Fn-111 (hereafter NgFn1,2) consists of two structural motifs of Fn-111, which are arranged in series at an obtuse angle with respect to each other (Figure 3A). Although a disulfide bond connects the two panels of the J3 domain of Fn-111, it has no other relation to the type of disulfide bond linkage. The function, if any, of a comparable loop in Fn-111 modules of neuronal adhesion molecules is unknown.
The F to G loop is the only loop that is spatially and structurally similar in the two domains of the NgFn1 ,2 structure. The relative orientation of the two Fn-111 domains in the crystal structure of NgFn1,2 is likely to be the same as would be observed in solution for the following reasons. The line indicates the position of the approximate 2-fold helix axis that relates the two domains.
We have described the structure of two N-terminal Fn-111 repeats (NgFn1,2) from Drosophila neuroglian, a neural CAM. The relative orientation of the two neuroglian Fn-111 domains is stabilized by the binding of a sodium ion at the interface between the domains. Also, binding of the immunoglobulin superfamily member contactin to the Fn-111 domains of tenascin is disrupted by the addition of three additional Fn-111 motifs that exist in an alternatively spliced isoform (Zisch et al., 1992).
Th1s neuroglian secretion plasmid was used to create a second plasmid for the secretion of the Fn-111 repeats. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Different extracellular domains of the neural cell adhesion molecule (N-CAM) are involved in different functions.
REFINEMENT OF THE STRUCTURE: A DETAILED
Crystallographic refinement begins as soon as an initial model of the structure is completed. These maps also highlight errors in the model, but do so in the context of the rest of the molecule's electron density. Thus, a Ramachandran plot of the entire protein is a graphical representation of the overall quality of the structure.
In the first cycle of the first refinement experiment, the initial model was minimized with simulated annealing using data from 6 to 2.1 A. The stereochemistry of the model was examined with PROCHECK and found to be reasonable (Figure 3-1. These plots show overall reliability of the placement of side chains in the model in terms of chi-1 and chi-2 angles.
The stereochemistry of the model was analyzed with PROCHECK and several residues with energetically unfavorable side chain conformations were examined. Thirty ordered water molecules were added to the +4cr [Fo-Fc] difference density peaks, and the sulfate anion near His646 was placed back into the model because the orientation of the oxygen atoms was clear. Minimization produced a significant decrease in Rfree, indicating that the information content of the model was improved.
The B value of the ion in the refined model was set to the average B value of its oxygen ligands, and the occupancy of the Na + ion was refined to 77%. Analysis of the geometry of the model confirmed that the main chain and side chain stereochemistry are reasonable. The refined native coordinates, with the B value of the ion set to the average ligand B value, were used in all occupancy calculations.
There was also a difference in the quality of the crystals from which the data was collected. Many of the crystals used in the soaking experiments were smaller and older than the crystal used to collect the native data. The refined occupancy of aLi+ ion in the native structure is 400%, showing that the majority of the bound ion is Na+.
None of the images in Ng's photomicrographs were clearly identified as a straight molecule. These experiments showed that the subclass identity of immunoglobulin CH2 domains modulates FcγRI binding.
REPEATS OF DROSOPHILA NEUROGLIAN
CHARACTERIZATION OF THE EXTRACELLULAR
As described in Chapter 2, the structure of the first two repeats of type lli fibronectin from Drosophila neuroglian (NgFnl,2) was used to model five tandem repeats of type lli fibronectin (NgFnl-5) in the whole neuroglian. Of the ten interdomain interfaces between tandem domains in the extracellular part of Ng, only the interface between the first two repeats of Fniii, NgFnl and NgFn2, has been structurally characterized. It has also been suggested that a high degree of flexibility would facilitate the diffusion of the binding sites of the molecule in the extracellular environment, thereby improving the binding kinetics6.
NgFnl-5 molecules with two bends can be divided into two groups: 'cis' proteins with both arms on the same side of the middle segment (Figure 5-2E) and 'trans' molecules with arms on opposite sides of the middle segment (Figure 5-2F ). Flash freezing eliminated radiation decay, but no improvements in crystal diffraction limits were observed (A. Huber, unpublished results). To facilitate the solution of the low-resolution FeRn-Fe crystal structure and to better describe the function of FeRn, we biochemically determined the stoichiometry of the FcRn-IgG complex.
Since it was not clear that the weakly bending complex crystals could be resolved, a study of the relative affinity of FeRn for different IgG molecules was undertaken. This type of analysis can identify high affinity complexes for crystallization and can lead to the localization of the ligand binding site. Information regarding the location of the FeRn binding site on IgG will facilitate the resolution of a low resolution complex structure.
Chimeric IgG molecules were produced by domain swapping between human IgG3 and IgG2, and the relative affinities of the chimeric molecules for FcγRI were determined. Investigation of the affinity of FeRn for different immunoglobulins could identify promising candidates for chimera studies and could also lead to the localization of the FeRn binding site through sequence comparisons. As previously mentioned, such localization of the FeRn binding site would not only better characterize the function of FeRn, but could lead to the crystallization of physiologically relevant FeRn complexes and the Fe fragment.
The results of the polyclonal affinity tests could not be used for a sequence-based localization of the FeRn binding site, because the polyclonal IgG contains a variety of IgG subclasses. The competitor concentration required to inhibit binding of the labeled rat IgG2a by 50 percent (l50) was estimated by plotting percent inhibition versus log (competitor concentration, nM) for each inhibitor IgG. An increase in complex stability coupled with changes in the amino acid sequence of the Fe could lead to new and better FcRn-Fc cocrystals.
