Adriana Huerta-Viga

Amplified Vibrational Circular Dichroism as a Probe of Local Biomolecular Structure

We show that the VCD signal intensities of amino acids and oligopeptides can be enhanced by up to 2 orders of magnitude by coupling them to a paramagnetic metal ion. If the redox state of the metal ion is changed from paramagnetic to diamagnetic the VCD amplification vanishes completely. From this observation and from complementary quantum-chemical calculations we conclude that the observed VCD amplification finds its origin in vibronic coupling with low-lying electronic states. We find that the enhancement factor is strongly mode dependent and that it is determined by the distance between the oscillator and the paramagnetic metal ion. This localized character of the VCD amplification provides a unique tool to specifically probe the local structure surrounding a paramagnetic ion and to zoom in on such local structure within larger biomolecular systems.

Protein Denaturation with Guanidinium: A 2D-IR Study.

Guanidinium (Gdm+) is a widely used denaturant, but it is still largely unknown how it operates at the molecular level. In particular, the effect of guanidinium on the different types of secondary structure motifs of proteins is at present not clear. Here, we use two-dimensional infrared spectroscopy (2D-IR) to investigate changes in the secondary structure of two proteins with mainly α-helical or β-sheet content upon addition of Gdm-13C15N3·Cl. We find that upon denaturation, the β-sheet protein shows a complete loss of β-sheet structure, whereas the α-helical protein maintains most of its secondary structure. These results suggest that Gdm+ disrupts β-sheets much more efficiently than α-helices, possibly because in the former, hydrophobic interactions are more important and the number of dangling hydrogen bonds is larger.

Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding

Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu– and Arg+ are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.

The structure of salt bridges between Arg+ and Glu- in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries

Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu(-)) and arginine (Arg(+)) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu(-) and Arg(+), which provide a sensitive structural probe of Glu(-)⋯Arg(+) salt bridges. We use this probe to investigate a β-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the β-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution.

A salt-bridge structure in solution revealed by 2D-IR spectroscopy

Salt bridges are known to be important for the stability of protein conformation, but up to now it has been difficult to study their geometry in solution. Here we characterize the spatial structure of a model salt bridge between guanidinium (Gdm) and acetate (Ac−) using two-dimensional vibrational (2D-IR) spectroscopy. We find that as a result of salt bridging the infrared response of Gdm and Ac− change significantly, and in the 2D-IR spectrum, salt bridging of the molecules appears as cross peaks. From the 2D-IR spectrum we determine the relative orientation of the transition-dipole moments of the vibrational modes involved in the salt bridge, as well as the coupling between them. In this manner we reconstruct the geometry of the solvated salt bridge. The stabilization of molecular conformations by the attractive interaction between oppositely charged ions (salt bridges) is of great relevance in many areas of science. In particular, biological systems such as proteins, often contain salt bridges between ionic side chains that determine their structure [1] and function. [2] It is therefore fundamental to characterize the solvated structure of salt-bridged ion pairs, but this is unfortunately not possible with conventional methods like NMR. In this communication we report the study of a biologicallyrelevant ion pair, that formed by guanidinium (Gdm) and acetate (Ac−), using two-dimensional infrared (2DIR) spectroscopy. This ion pair is a model for salt bridges between an arginine and a carboxylate side group (from glutamate or aspartate), which occur commonly in proteins. [3] The molecular structure of this ion pair is shown in Fig. 1A. Isolated Gdm has D3 symmetry and a degenerate mode at 1600 cm−1 due to a combined CN3 antisymmetric stretch and NH2 scissors motion. [4] This degeneracy is also observed in aqueous solution, but it is broken in viscous solvents. [5] When dissolving deuterated Gdm (guanidine·DCl, >98% purity) in deuterated dimethylsulfoxide (DMSO), we observe a similar splitting between the frequencies of the two CN3D + 6 modes, as can be seen in Fig. 1B. In the following, we will refer to the highand low-frequency CN3D + 6 of Gdm + as the Gdm+HF and Gdm+LF modes, respectively. Interestingly, when an equimolar amount of Ac− ions is added to the solution (guanidine acetate salt, >98% purity), this splitting becomes larger. It is known that Gdm and Ac− have a strong binding affinity in DMSO, forming more than 98% dimers at the concentration used in our experiments. [6] This suggests that the larger splitting between the Gdm modes is due to an interaction with the Ac− ion. Moreover, Ac− (tetrabutylammonium acetate, >97% purity) has an absorption band at 1580 cm−1 in DMSO (shown in Fig. 1B) due to the COO− antisymmetric stretch mode. This mode red-shifts after dimerization with Gdm. The change in the infrared response of both the Gdm and the Ac− ions upon dimerization strongly suggests that there is a coupling between the vibrational modes of these two molecules. The 2D-IR spectrum of Gdm · · ·Ac− confirms unambiguously that the Gdm+HF and Gdm + LF modes are both coupled to the COO− stretch mode of Ac−. We use a femtosecond pump-probe setup that has been described elsewhere. [7] The resulting spectra are shown in Figs. 2B and C for parallel and perpendicular polarization of the pump and probe pulses, respectively. The non-zero offdiagonal response in the 2D-IR spectrum indicates that there is a coupling between the two CN3D + 6 modes of Gdm and, more importantly, between each of them and the COO− stretch mode of Ac−. These cross peaks can be seen better in slices along both the pump and probe axes of the 2D-IR spectra. Fig. 3A shows a cross section for νpump = νCOO− for parallel and perpendicular polarization of the pump and probe pulses. In the cross section, the cross peaks between the COO− stretch mode and each of the two CN3D + 6 modes are clearly visible. Note that the diagonal response of the COO− stretch FIG. 1. (A) Molecular structure of the Gdm · · ·Ac− dimer obtained using ab initio methods. The corresponding transition-dipole moments of the COO− stretch mode (1) and of the CN3D + 6 low and high frequency modes (2 and 3, respectively) are indicated by arrows. (B) Infrared absorption spectrum of Gdm· · ·Ac−, Gdm and Ac− in DMSO (400 mM, solvent subtracted). Shifts of the COO− and highfrequency CN3D + 6 bands are indicated by arrows. 1 ar X iv :1 30 1. 34 15 v1 [ ph ys ic s. ch em -p h] 1 5 Ja n 20 13