Dynamics of DNA molecules in gel studied by fluorescence microscopy.

The dynamics of individual DNA molecules in a thin gel were studied with fluorescence microscopy. Driven by an electric field, molecules hooked around isolated obstacles and became extended. By analyzing molecular images, we identified the reptation tube and primitive chain. When the field was turned off, the molecules relaxed. The relaxation time tau1 and primitive chain length at equilibrium depend on N, the size of the molecule in base pairs, consistently with reptation theory. Using five yeast chromosomal DNAs ranging in size from 245 kb to 980 kb, we found that: These results constitute a way of sizing individual DNA molecules by imaging rather than by gel electrophoresis.

Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected].

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

Use of global amino acid replacements to define the requirements for MHC binding and T cell recognition of moth cytochrome c (93-103).

Substitution with all naturally occurring L-amino acids at each of 11 residues of the IEk-restricted month cytochrome c (93-103) epitope has allowed us to analyze the requirements for MHC binding and T cell recognition to a level of definition not previously possible. Substitutions at only three positions systematically affect MHC binding and three others appear to be the major TCR contacts. Interestingly, changing residues involved in MHC binding can ablate T cell recognition without altering MHC association. Additionally, residue identity at two positions that do not appear critical for MHC binding, nor to be involved in specific T cell contact, nonetheless dramatically affect T cell responses. This suggests that peptides differing only slightly in sequence can have significantly altered conformations within the class II MHC binding groove. We have also developed a simple scoring program that uses the binding data to quantitate how well a given peptide fits the MCC motif. All strongly immunogenic IEk-restricted epitopes score highly (> or = 0.70, where 1.0 is perfect concordance), and only 3% of all potential nonameric peptides in the two main protein sequence databases have scores greater than 0.70. This indicates that the global amino acid replacement approach using a single peptide is an efficient means of deriving binding motifs for a given class II MHC molecule, and should aid in the identification of novel T cell epitopes.

pH affects both the mechanism and the specificity of peptide binding to a class II major histocompatibility complex molecule.

We have compared the contribution of electrostatic forces in the binding of antigenic peptides to the class II MHC molecule, IEk, at weakly acidic (pH 5.4) and neutral (pH 7.5) pH values. The binding of specific moth cytochrome c (MCC) and hemoglobin (Hb) peptides to IEk is very sensitive to ionic strength at pH 7.5 but not at pH 5.4, indicating that the mechanism of peptide binding is pH-dependent. Substitution of the C-terminal Lys in MCC for an Ala residue selectively destroyed peptide binding at neutral pH and increased the dissociation rate at least 30-fold, implicating this residue in the pH-dependent electrostatic interaction. The presence of a C-terminal Lys in many of the peptides that are restricted to IEk suggests that this electrostatic interaction is widely used to bind peptides to this MHC molecule. We also probed the electrostatic environment of the peptide binding groove adjacent to the N-terminus of the bound peptide by rapid-diffusion fluorescence energy transfer using a terbium-labeled MCC peptide. In this region of the peptide binding groove, more negative charge is present at pH 7.5 than at pH 5.4. These findings indicate the importance of MHC carboxylates to the mechanism and specificity of peptide binding. The biological importance of having two distinct mechanisms of peptide binding at different pH may be that it acts to broaden the spectrum of antigenic peptides that can be presented to T-cells.