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We have developed a method (hygroscopic desorption) for measuring the binding of small molecules to membranes. With this method, we have found that the binding of the amphipathic compounds chlorpromazine, 2,4-dinitrohenol, and 1-decanol to various cell membranes is remarkably Fow, with partition coefficients, Kp, no larger than about 0.1.On the other hand, with phospholipid vesicles of large or small diameters, Kp values for these compounds were much larger.The results suggest that there exists in membranes a large internal pressure that excludes the amphipaths from the membranes and that does not exist in phospholipid vesicles. It is widely accepted that small amphipathic molecules (amphipaths) are extensively solubilized in the lipid bilayer portions of most biological membranes. This is presumed to occur by insertion of the hydrophobic portion of an amphipath into the fatty acyl chain regions of the bilayer, whereas the hydrophilic end is situated at the membrane-water interface. This picture has emerged from various sources, but, in particular, from (Amersham). '251-Labeled concanavalin A (86 Ci/mmol; 1 Ci = 3.7 X 10'0 becquerels) was prepared from affinity-purified concanavalin A by the chloramine-T method (3). ['4C]Dipalmitoyl phosphatidylcholine was a gift from Raoul Bejar.Solvent-cast polycarbonate films were obtained as large, thin sheets 2-3 ,um thick from Mobay Chemicals (Pittsburgh, PA) or 5-10 ,um thick from Nuclepore and prepared for use as described below. Glass spacer filters (47 cm in diameter and 100 ,m thick) and bleached cellulose pads (37 cm in diameter and 0.4 cm thick) were obtained from Schleicher and Schuell, and Millipore, respectively.Erythrocytes and Lymphoma Cells. Erythrocytes were obtained on the day of an experiment from normal donors. Peripheral blood, drawn into acid/citrate/dextrose, was washed three times in isotonic Tris/saline (140 mM NaCl/20 mM Tris-HCl, pH 6.4) at 4°C and the intact cells were harvested by aspiration from under the "buffy" coat. Erythrocyte ghosts were prepared from these cells by lysis in 20 mM Tris-HCl (pH 7.4) at 4°C and by washing five times in 30 vol of this buffer.The lymphoma cells used were a T cell line (S49-1TB-2-3) and a B cell line (S194/5-XXO-BU-1), cultured in Dulbecco's modified Eagle's medium and 10% fetal calf serum, which were obtained from R. Hyman of the Salk Institute for Biological Studies. Immediately prior to use, the cells were washed three times in isotonic phosphate-buffered saline (P1/NaCl) at pH 7.4. In all of the suspensions used, more than 99% of the cells were impermeable to trypan blue.Lymphoma 'ghost' membranes were prepared from washed cells by lysis in 30 vol of 20 mM P1/NaCl (pH 8.0) containing 0.1 mM CaCl2, to which was added staphylococcal deoxyribonuclease (Worthington) at a final concentration of 20 ,g of protein per ml of lymphoma cells. After incubation at 40C for 45 min, the digest was centrifuged for 10 min at 6000 X g and the pellet was washed three times in 30 vol of calcium-free Abbreviation: Pi/NaCl...

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By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (residues 95-203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome. After expression in Escherichia coli, two phenotypic assays were performed. The first assay tested for RNA-dependent DNA polymerase activity. The other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa subunit. The resulting phenotypic data provided a ''genetic'' means to identify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface. Several HIV-1 RT crystal structures were used to evaluate the mutational analysis. Our genetic map correlates well with the crystal structures. Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues. The important functional residues are found near the enzyme active site. Many residues important for the stability of the RT participate in potential hydrogen bonding or hydrophobic interactions in the protein interior. In addition to providing a better understanding of the HIV-1 RT, this work demonstrates the utility of saturation mutagenesis to study the function, structure, and stability of proteins in general. This strategy should be useful for studying proteins for which no crystallographic data are available.The development of oligonucleotide-directed mutagenesis (1) enhanced the power of genetic analysis by providing a method to introduce a mutation at a specific position in a DNA sequence. This technique may be used to make a specific amino acid substitution in a protein by altering codons. An adaptation of site-specific mutagenesis led to saturation mutagenesis (reviewed in ref. 2), which provides an approach to make libraries containing a collection of mutations at every base in a DNA element (3) or at every amino acid in a protein (4).X-ray crystallography is a powerful tool for studying protein function and structure. However, the use of this method is limited to proteins that crystallize. With the constant discovery of new proteins with biological and medical importance, additional methods that provide functional and structural information are extremely valuable. In this study, we assess the potential of saturation mutagenesis for the study of function and stability of proteins for which structural data are difficult to obtain, and as a means to offer additional insight into proteins whose structures are known. By using saturation mutagenesis we collected 366 unique single missense mutants in a 109-aa region (residues P95 through E203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT).The HIV-1 RT is encoded by the viral pol gene, which contains the coding sequences for the protease, R...