THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION

During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.

Sequence and Structure Based Protein Folding Studies With Implications

As the expression of the genetic blueprint, proteins are at the heart of all biological systems. The ever increasing set of available protein structures has taught us that diversity is the hallmark of their architecture, a fundamental characteristic that enables them to perform the vast array of functionality upon which all of life depends. This diversity, however, is central to one of the most challenging problems in molecular biology: how does a folding polypeptide chain navigate its way through all of the myriad of possible conformations to find its own particular biologically active form? With few overarching structural principles to draw upon that can be applied to all protein architecture, the search for a solution to the protein folding problem has yet to produce an algorithm that can explain and duplicate this fundamental biological process. In this thesis, we take a two-pronged approach for investigating the protein folding process. Our initial statistical studies of the distributions of hydrophobic and hydrophilic residues within α-helices and β-sheets suggest (i) that hydrophobicity plays a critical role in helix and sheet formation; and (ii) that the nucleation of these motifs may result in largely unidirectional growth. Most tellingly, from an examination of the amino acids found in the smallest β-sheets, we do not find any evidence of a β-nucleating code in the primary protein sequence. Complementing these statistical analyses, we have analyzed the structural environments of several ever-widening aspects of protein topology. Our examination of the gaps between strands in the smallest β-sheets reveals a common organizational principle underlying β-formation involving strands separated by large sequential gaps: with very few exceptions, these large gaps fold into single, compact structural modules, bringing the β-strands that are otherwise far apart in the sequence close together in space. We conclude, therefore, that β-nucleation in the smallest sheets results from the co-location of two strands that are either local in sequence, or local in space following prior folding events. A second study of larger β-sheets both corroborates and extends these findings: virtually all large sequential gaps between pairs of β-strands organize themselves into an hierarchical arrangement, creating a bread-crumb model of go-and-come-back structural organization that ultimately juxtaposes two strands of a parental β-structure that are far apart in the sequence in close spatial proximity. In a final study, we have formalized this go-and-come-back notion into the concept of anti-parallel double-strandedness (DS), and measure this property across protein architecture in general. With over 90% of all residues in a large, non-redundant set of protein structures classified as DS, we conclude that DS is a unifying structural principle that underpins all globular proteins. We postulate, moreover, that this one simple principle, anti-parallel double-strandedness, unites protein structure, protein folding and protein evolution.