Agents that target the two highly conserved Zn fingers of the human immunodeficiency virus (HIV) nucleocapsid p7 (NCp7) protein are under development as antivirals. These agents covalently modify Zn-coordinating cysteine thiolates of the fingers, causing Zn ejection, loss of native protein structure and nucleic acid binding capacity, and disruption of virus replication. Concentrations of three antiviral agents that promoted in vitro Zn ejection from NCp7 and inhibited HIV replication did not impact the functions of cellular Zn finger proteins, including poly(ADP-ribose) polymerase and the Sp1 and GATA-1 transcription factors, nor did the compounds inhibit HeLa nuclear extract mediated transcription. Selectivity of interactions of these agents with NCp7 was supported by molecular modeling analysis which (1) identified a common saddle-shaped nucleophilic region on the surfaces of both NCp7 Zn fingers, (2) indicated a strong correspondence between computationally docked positions for the agents tested and overlap of frontier orbitals within the nucleophilic loci of the NCp7 Zn fingers, and (3) revealed selective steric exclusion of the agents from the core of the GATA-1 Zn finger. Further modeling analysis suggests that the thiolate of Cys49 in the carboxy-terminal finger is the site most susceptible to electrophilic attack. These data provide the first experimental evidence and rationale for antiviral agents that selectively target retroviral nucleocapsid protein Zn fingers.

A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.

In the last few years there has been an increased effort to develop technologies capable of identifying and quantifying large numbers of proteins expressed within a cell system (i.e., the proteome). The complexity of the mixtures being analyzed has made the development of effective fractionation and separation methods a critical component of this effort. This review highlights many of the protein and peptide fractionation and separation methods, such as electrophoresis and high-performance liquid chromatography (HPLC), which have experienced significant development over the past forty years. Modern instrumental strategies for the resolution of cell proteins, based on separations employing a single high-resolution or multidimensional approach, and the relative merits of each, will be discussed. The focus of this manuscript will be on the development of multidimensional separations such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), HPLC/HPLC, and HPLC-capillary electrophoresis and their application to the characterization of complex proteome mixtures.

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