Rad51 protein (Rad51) is central to recombinational repair of double-strand DNA breaks. It polymerizes onto DNA and promotes strand exchange between homologous chromosomes. We visualized the real-time assembly and disassembly of human Rad51 nucleoprotein filaments on double-stranded DNA by single-molecule fluorescence microscopy. Rad51 assembly extends the DNA by approximately 65%. Nucleoprotein filament formation occurs via rapid nucleation followed by growth from these nuclei. Growth does not continue indefinitely, however, and nucleoprotein filaments terminate when approximately 2 mum in length. The dependence of nascent filament formation on Rad51 concentration suggests that 2-3 Rad51 monomers are involved in nucleation. Rad51 nucleoprotein filaments are stable and remain extended when ATP hydrolysis is prevented; however, when permitted, filaments decrease in length as a result of conversion to ADP-bound nucleoprotein complexes and partial protein dissociation. Dissociation of Rad51 from dsDNA is slow and incomplete, thereby rationalizing the need for other proteins that facilitate disassembly.
A characterization of the conformation and stability of model peptide systems that form β-sheets in aqueous solutions is considerably important in gaining insights into the mechanism of β-sheet formation in proteins. We have characterized the conformation and equilibrium folding and unfolding of two 20-residue peptides whose NMR spectra suggest a three-stranded β-sheet topology in aqueous solution: Betanova [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253−256] and DPDP with d-Pro-Gly segments at the turns [Schenck, H. L., and Gellman, S. H. (1998) J. Am. Chem. Soc. 120, 4869−4870]. Both circular dichroism (CD) and infrared measurements indicate only 20−26% β-sheet-like structure at 5 °C for Betanova and 42−59% β-sheet for DPDP. For both peptides, the CD and infrared spectra change nearly linearly with increasing temperatures (or urea concentrations) and lack a sigmoidal signature characteristic of cooperative unfolding. Fluorescence resonance energy transfer (FRET) measurements between donor and acceptor molecules attached to the two ends confirm that Betanova is largely unstructured even at 10 °C; the average end-to-end distance estimated from FRET is closer to that of a random coil than a structured β-sheet. In DPDP, the FRET results indicate a more compact structure that remains compact even at high temperatures (∼80 °C) or high urea concentrations (∼8 M). These results indicate that both these peptides access an ensemble of conformations at all temperatures or denaturant concentrations, with no significant free energy barrier separating the "folded" and "unfolded" conformations.
In this contribution we report optical spectroscopic data on a series of designed β hairpins previously shown by NMR to contain a substantial population of β-sheet structure. These models contain a designed hydrophobic cluster and a dPro-Gly sequence to promote formation of a turn geometry. FTIR, electronic and vibrational CD (ECD and VCD) spectra for these small peptides are comparable to expected bandshapes for peptides of high β-sheet content. The dPro-Gly sequence provides a better turn motif than Asn-Gly as measured by its β-sheet spectral characteristics. IR and VCD spectra are in qualitative agreement with theoretical simulations based on transfer of parameters from ab initio quantum mechanical force field and intensity computations for the turn and strands. These calculations provide assignments for some distinguishing modes in both IR and VCD spectra. Increased sheet structure can be induced in these hairpins by use of mixed solvent conditions. Thermal denaturation studies reveal that these hairpins undergo very broad unfolding transitions. Guanidine hydrochloride unfolding transitions for the selected hairpin models are similarly broad. However, the "end-states" of temperature and chaotropic denaturation are spectroscopically differentiable.
The DNA sequence of the chicken glutamine synthetase gene contains an A·T-rich stretch of approximately 1500 base pairs in the 3' noncoding regions of exon 7 [Pu, H., & Young, A. P. (1989) Gene 18, 169−175]. Within this region several palindromic sequences occur that could conceivably form intramolecular structures. One such perfect inverted repeat sequence resides between positions 2605 and 2623. To investigate the hairpin-forming potential for this sequence, optical and calorimetric melting and gel electrophoresis studies have been performed on the following synthetically prepared DNA and RNA oligomer subsequences: DNA, 5'd-T-T-T-T-T-T-A-A-T-A-A-T-T-A-A-A-A-A-A-3'; and RNA, 5'r-U-U-U-U-U-U-A-A-U-A-A-U-U-A-A-A-A-A-A-3'. The DNA strand corresponds to the coding strand sequence while the RNA strand represents the transcribed mRNA. Results of melting analysis of these 19-base, partially self-complementary strands performed in 115 mM Na+ yielded evaluations of their thermodynamic transition parameters. These values are consistent with the melting of unimolecular structures, presumably hairpins. Thermodynamic parameters evaluated by analysis of the optical melting transitions assuming a two-state model and measured directly by differential scanning calorimetry agreed within experimental error. Therefore, melting behavior of the hairpins is all-or-none like. The DNA hairpin is slightly more stable than the RNA hairpin with melting enthalpy ΔH0 = 41.2 ± 3.8 kcal/mol and entropy ΔS0 = 133 ± 11 cal/K·mol (eu) compared to ΔH0 = 32.0 ± 6.0 kcal/mol and entropy ΔS0 = 105 ± 20 eu for the RNA. Gel electrophoretic analysis of these oligomers alone and in various mixtures with their DNA and RNA complementary strands was also performed. Consistent with interpretations of melting results, these experiments revealed both strands alone preferentially form intramolecular hairpin structures. In mixtures in which their complementary strands are in vast molar excess (stoichiometric ratios > 10:1), the intramolecular structures are converted to intermolecular duplexes. For the DNA and RNA strands examined, the conversion is not complete until over a 1000-fold excess of the complementary strand is added. Semiquantitative analysis of gel electrophoretograms enabled evaluations of the relative free energies of the hairpin and duplex states as a function of complementary strand concentration. With the finding that these sequences preferentially form hairpins, potential roles these structures could play in regulatory activities are considered.
Model β-hairpin peptides based on variations in the turn sequence of Cochran's tryptophan zipper peptide, SWTWENGKWTWK, were studied using electronic circular dichroism (ECD), fluorescence, and infrared (IR) spectroscopies. The trpzip2 Asn-Gly turn sequence was substituted with Thr-Gly, Aib-Gly, (D)Pro-Gly, and Gly-Asn (trpzip1) to study the impact of turn stability on β-hairpin formation. Stability and conformational changes of these hairpins were monitored by thermodynamic analyses of the temperature variation of both FTIR (amide I') and ECD spectral intensities. These changes were fit to a two-state model which yielded different T(m) values, representing the folding/unfolding process, for hairpins with different β-turns. Different β-turns show systematic contributions to hairpin structure formation, and their inclusion in hairpin design can modify the folding pathways. Aib-Gly or (D)Pro-Gly sequences stabilize the turn resulting in residual Trp-Trp interaction at high temperatures, but at the same time the β-structure (cross strand H-bonds) can become less stable due to constraints of the turn, as seen for (D)Pro-Gly. The structure of the Aib-Gly turn containing hairpin was determined by NMR and was shown to be like trpzip2 (Asn-Gly turn) as regards turn and strand geometries, but to differ from trpzip1 (Gly-Asn turn). The Munoz and Eaton statistical mechanically derived multistate model, tested as an alternate point of view, represented contributions from H-bonds and hydrophobic interactions as well as conformational change as interdependent. Use of different spectral methods that vary in dependence on these physical interactions along with the structural variations provided insight to the complex folding pathways of these small, well-folded peptides.
