SGER: Molecular Analysis of Connexin pI Variants

Yeshiva University, New York NY

Investigators

Abstract

Covalent modification of proteins is a well-established mechanism by which their structure and properties are altered to accommodate biological function. Principals elucidated for soluble proteins apply to integral membrane proteins as well, although many underlying molecular features in this class of proteins are less well established than for soluble proteins. The vast majority of plasma membrane integral membrane proteins are glycosylated and most are phosphorylated, modifications that introduce anionic groups. Hence, even when analyzed successfully by techniques such as isoelectric focusing and two-dimensional electrophoresis, integral membrane proteins often exhibit pIs significantly more acidic than those anticipated based upon their primary structure, now mostly deduced from the sequence of their cDNAs. Recent evidence from Dr. Hertzberg's laboratory has demonstrated that several connexins, unglycosylated integral membrane proteins comprising gap junctions, have pI variants far more acidic than can be accounted for by phosphorylation or other known modifications. The modification altering the charge of these proteins does not significantly alter their mass as detected by relatively inexact methods such as SDS-PAGE. Furthermore, it appears as though connexin modification occurs during its trafficking and is likely to occur at distinct cytoplasmic compartments through which connexins pass on their way to assembly into gap junctions in the plasma membrane. Establishing the underlying molecular mechanism responsible for alteration of connexin pIs is the key focus of this project. Mass spectrometry has emerged as the method of choice for elucidation of the molecularbasis of protein covalent modification and determination the modified residues. While notable progress has been made in recent years in the biochemical analysis of membrane proteins, including crystallographic analysis of a few, their manipulation for many types of analyses is limited by their insolubility in organic solvents or requiring the use of detergents incompatible with subsequent analyses. In the case of connexins, these difficulties are even more profound: success in their initial isolation and characterization was dependent on their insolubility in detergents. This property has compounded the difficulty of their analysis by state of the art techniques such as mass spectrometry. Preliminary experiments have established approaches to peptide purification and mass spectroscopic analysis that will serve as the basis for analysis of the modification(s) altering connexin pIs. This project is driven by efforts to identify and characterize the (novel?) low molecular weight covalent modification of connexins, a line of exploratory research that will have significant implications for understanding of the structure and function of membrane proteins. It may be that this modification, which preliminary experiments indicate is not likely to be restricted to connexins, has been overlooked because the presence of pI variants of other membrane proteins would have been attributed to phosphorylation or the presence of acidic sugars (e.g. sialic acid). Identification of the modification altering connexin pIs will permit evaluation of its role in trafficking, assembly and function of gap junction channels. Depending upon the position of the negative charges, models of the structure and function of selectivity filters, regulatory sites and the channels themselves may require rethinking. Hence, studies undertaken as part of this project may prove to be of general significance in proteome analysis.