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Date of Award

2004

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Patrick Jones

First Committee Member

Arnold Falick

Second Committee Member

David Sparkman

Third Committee Member

Craig Vierra

Fourth Committee Member

Andreas Franz

Fifth Committee Member

Jianhua Ren

Abstract

Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In this study we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus , whose DNA coding sequences have not been reported. Based upon solubility differences in 8 M guanidine hydrochloride, it is demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining that the egg case silk is relatively complex at the molecular level, containing a large number of proteins with differing molecular weights. Protein components of egg case silk with a size about 100 kDa were obtained by a solubilization time course study, which indicates these proteins are likely to be embedded in the silk filament. Peptides in these 100 kDa proteins were released by tryptic in-gel and in-solution digestion. The peptides were sequenced using a MALDI tandem TOF mass spectrometer. Some of the de novo sequences were confirmed using a linear ion trap mass spectrometer equipped with a nanospray ion source. Combining the peptide sequences obtained, reverse genetics was employed to trace silk genes encoding proteins containing these de novo peptides. Three silk protein coding sequences were successfully discovered, which encode silk proteins named 3B, T1 and ECSP-1, respectively. 3B and T1 show the standard fibroin protein pattern. Amino acid repeat patterns were observed in these two silk clones. But the amino acid compositions of 3B and T1 show differences with the total amino acid composition of egg case silk, and also, the peptide sequences cannot be found in the primary amino acid sequences of 3B and T1. ECSP-1 protein represents one of the egg case silk proteins with a size of about 100 kDa. A number of peptide sequences obtained by mass spectrometric de novo sequencing were successfully located in ECSP-1's primary amino acid sequence. Sequence analysis demonstrates ECSP-1 represents a new class of silk proteins, with fibroin-like properties. The expression pattern of ecsp-1 is largely restricted to the tubuliform gland inside of the L. hesperus spider, with lower levels detected in the major and minor ampullate glands, which also confirms the identity of ECSP-1. It is also demonstrated that ECSP-1 assembles into higher aggregate structures through the formation of disulfide bonds. Peptide sequences from silk proteins from the Tarantula spider Grammostola rosea were also obtained. These sequences will be beneficial in obtaining genes encoding the silk from this spider species.

Pages

177

ISBN

9780496117895 , 0496117890

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