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.

Conformational changes in single-strand DNA as a function of temperature by SANS.

Small-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5'-ATGCTGATGC-3', in sodium phosphate buffer solution at 10 degrees C temperature increments from 25 degrees C to 80 degrees 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 degrees C to 43 degrees C, an expansion in the pitch direction with a slight decrease in the diameter from 43 degrees C to 53 degrees C, and finally a dramatic increase in the pitch and diameter from 53 degrees C to 80 degrees 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 degrees C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60 +/- 3 kJ mol(-1) that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47 +/- 1 degrees 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.

A coarse graining approach to determine nucleic acid structures from small angle neutron scattering profiles in solution.

We present a theoretical method to calculate the small angle neutron scattering profile of nucleic acid structures in solution. Our approach is sensitive to the sequence and the structure of the nucleic acid. In order to test our approach, we apply this method to the calculation of the experimental scattered intensity of the decamer d(CCAACGTTGG)2 in H2O. This sequence was specifically chosen for this study as it is believed to adopt a canonical B-form structure in 0.3 M NaCl. We find that not only will our methodology reproduce the experimental scattered intensity for this sequence, but our method will also discriminate between B-, A- and Z-form DNA. By studying the scattering profile of this structure in 0.5 and 1.0 M NaCl, we are also able to identify tetraplex and other similar oligomers formation and to model the complex using the experimental scattering data in conjunction with our methodology.

Experimental and computational studies of the desensitization process in the bovine rhodopsin-arrestin complex.

The deactivation of the bovine G-protein-coupled receptor, rhodopsin, is a two-step process consisting of the phosphorylation of specific serine and threonine residues in the cytoplasmic tail of rhodopsin by rhodopsin kinase. Subsequent binding of the regulatory protein arrestin follows this phosphorylation. Previous results find that at least three phosphorylatable sites on the rhodopsin tail (T340) and at least two of the S338, S334, or S343 sites are needed for complete arrestin-mediated deactivation. Thus, to elucidate the details of the interaction between rhodopsin with arrestin, we have employed both a computational and an in vitro experimental approach. In this work, we first simulated the interaction of the carboxy tail of rhodopsin with arrestin using a Monte Carlo simulated annealing method. Since at this time phosphorylation of specific serines and threonines is not possible in our simulations, we substitute either aspartic or glutamic acid residues for the negatively charged phosphorylated residues required for binding. A total of 17 simulations were performed and analysis of this shows specific charge-charge interactions of the carboxy tail of rhodopsin with arrestin. We then confirmed these computational results with assays of comparable constructed rhodopsin mutations using our in vitro assay. This dual computational/experimental approach indicates that sites S334, S338, and T340 in rhodopsin and K14 and K15 on arrestin are indeed important in the interaction of rhodopsin with arrestin, with a possible weaker S343 (rhodopsin)/K15 (arrestin) interaction.

Vibrational spectroscopy and the development of new force fields for biological molecules.

The role of vibrational spectroscopy in the testing of force fields of biological molecules and in the determination of improved force fields is discussed. Analysis shows that quantitative testing of potential energy surfaces by comparison with spectroscopic data generally requires calculations that include anharmonic couplings between different vibrational modes. Applications of the vibrational self-consistent field (VSCF) method to calculations of spectroscopy of biological molecules are presented, and comparison with experiment is used to determine the merits and flaws of various types of force fields. The main conclusions include the following: (1) Potential surfaces from ab initio methods at the level of MP2 yield very satisfactory agreement with spectroscopic experimental data. (2) By the test of spectroscopy, ab initio force fields are considerably superior to the standard versions of force fields such as AMBER or OPLS. (3) Much of the spectroscopic weakness of AMBER and OPLS is due to incorrect description of anharmonic coupling between different vibrational modes. (4) Potential surfaces of the QM/MM (Quantum Mechanics/Molecular Mechanics) type, and potentials based on improved versions of semi-empirical electronic structure theory, which are feasible for large biological molecules, yield encouraging results by the test of vibrational spectroscopy.