Conformational Changes in Single-Strand DNA as a Function of Temperature by SANS

AbstractSmall-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5′-ATGCTGATGC-3′, in sodium phosphate buffer solution at 10°C temperature increments from 25°C to 80°C. Cylindrical, helical, and random coil shape models were fitted to the SANS measurements at each temperature. All the shapes exhibited an expansion in the diameter direction causing a slightly shortened pitch from 25°C to 43°C, an expansion in the pitch direction with a slight decrease in the diameter from 43°C to 53°C, and finally a dramatic increase in the pitch and diameter from 53°C to 80°C. Differential scanning calorimeter scans of the sequence in solution exhibited a reversible two-state transition profile with a transition temperature of 47.5±0.5°C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60±3kJ mol−1 that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47±1°C was also obtained from ultraviolet optical density measurements of strand melting scans of the single-strand DNA. This transition corresponds to unstacking of the bases of the sequence and is responsible for the thermodynamic discrepancy between its binding stability to its complementary sequence determined directly at ambient temperatures and determined from extrapolated values of the melting of the duplex at high temperature

Terahertz Spectroscopy of Bacteriorhodopsin and Rhodopsin: Similarities and Differences

We studied the low-frequency terahertz spectroscopy of two photoactive protein systems, rhodopsin and bacteriorhodopsin, as a means to characterize collective low-frequency motions in helical transmembrane proteins. From this work, we found that the nature of the vibrational motions activated by terahertz radiation is surprisingly similar between these two structurally similar proteins. Specifically, at the lowest frequencies probed, the cytoplasmic loop regions of the proteins are highly active; and at the higher terahertz frequencies studied, the extracellular loop regions of the protein systems become vibrationally activated. In the case of bacteriorhodopsin, the calculated terahertz spectra are compared with the experimental terahertz signature. This work illustrates the importance of terahertz spectroscopy to identify vibrational degrees of freedom which correlate to known conformational changes in these proteins