We describe the use of gel electrophoresis in studies of equilibrium binding, site distribution, and kinetics of protein-DNA interactions. The method, which we call protein distribution analysis, is simple, sensitive and yields thermodynamically rigorous results. It is particularly well suited to studies of simultaneous binding of several proteins to a single nucleic acid. In studies of the lac repressor-operator interaction, we found that binding to the so-called third operator site (03) is 15-18 fold weaker than operator binding, and that the binding reactions with the first and third operators are uncoupled, implying that there is no communication between the sites. Pseudo-first order dissociation kinetics of the repressor-203 bp operator complex were found to be temperature sensitive, with delta E of 80 kcal mol-1 above 29 degrees C and 26 kcal mol-1 below. The half life of the complex (5 min at 21 degrees C) is shorter than that reported for very high molecular weight operator-containing DNAs, but longer than values reported for much shorter fragments. The binding of lac repressor core to DNA could not be detected by this technique: the maximum binding constant consistent with this finding is 10(5) M-1.

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

The importance of the left-handed polyproline II (PPII) helical conformation has recently become apparent. This conformation generally is involved in two important functions: protein-protein interactions and structural integrity. PPII helices play vital roles in a variety of processes including signal transduction, transcription, and cell motility. Proline-rich regions of sequence are often assumed to adopt this structure. Remarkably, little is known about the physical determinants of this secondary structure type. In this study, we have explored the formation of PPII helices by a short poly(proline) peptide. In addition, the results from experiments used to determine the propensities for apolar residues, plus glycine, asparagine, and glutamine, to adopt this structure in a poly(proline)-based host peptide are reported here. Proline possesses the highest intrinsic propensity, with glutamine, alanine, and glycine having surprisingly high propensities.-Branched residues possess the lowest propensities of the residues examined. It is postulated that propensities possessed by apolar residues are due in part to peptide-solvent interactions, and that the remarkably high propensity possessed by glutamine may be due to a side chain to backbone hydrogen bond. These data are the first step toward a molecular understanding of the formation of this important, and yet little studied, secondary structure.In recent years, the left-handed polyproline II (PPII) 1 helical conformation has been elevated from the status of a relatively rare and seemingly uninteresting secondary structure to one that is surprisingly common and of the utmost importance. This structure plays a central role in numerous vital processes including signal transduction, transcription, cell motility, and the immune response. Proline-rich ligands of the cytoskeletal protein profilin (1), as well as those of the SH3, WW, and EVH1 protein interaction domains, are bound in this conformation (2). The peptide ligands of class II MHC molecules are also bound in the PPII conformation (3). PPII helices are major features of collagens (4) and plant cell wall proteins (5). The PPII helix is believed to be the dominant conformation for many proline-rich regions of sequence (PRRs) (6). Sequences not rich in proline also adopt this structure. For example, poly(lysine), poly(glutamate), and poly(aspartate) peptides form PPII helices (7). Around 2% of all residues in known protein structures are found in PPII helices at least four residues long (8, 9). As many as 10% of all residues are found in the PPII conformation, although not necessarily as part of PPII helices (10). PPII helices have also been hypothesized to be a major component of protein denatured states (11-14), giving them a role in a most fundamental process. Recently, Blanch et al. (15) have suggested that the PPII helix might be the precursor conformation in amyloid formation. Given the preceding, it is truly remarkable how little is known about the physical determinants of the PPII helical conformat...

Integrins are the major adhesion receptors of leukocytes and platelets. β 1 and β 2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin α IIb β 3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after 'inside-out' signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β 2 integrins because of mutations in the gene specifying the β 2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting α IIb β 3 integrin cause the bleeding disorder termed Glanzmann's thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann's thrombasthenia. Their hematopoietically-derived cells express β 1 , β 2 and β 3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects' lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration.