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Ishikawa, Haruto,Kim, Seongheun,Kwak, Kyungwon,Wakasugi, Keisuke,Fayer, Michael D National Academy of Sciences 2007 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.104 No.49
<P>Intramolecular disulfide bonds are understood to play a role in regulating protein stability and activity. Because disulfide bonds covalently link different components of a protein, they influence protein structure. However, the effects of disulfide bonds on fast (subpicosecond to approximately 100 ps) protein equilibrium structural fluctuations have not been characterized experimentally. Here, ultrafast 2D-IR vibrational echo spectroscopy is used to examine the constraints an intramolecular disulfide bond places on the structural fluctuations of the protein neuroglobin (Ngb). Ngb is a globin family protein found in vertebrate brains that binds oxygen reversibly. Like myoglobin (Mb), Ngb has the classical globin fold and key residues around the heme are conserved. Furthermore, the heme-ligated CO vibrational spectra of Mb (Mb-CO) and Ngb (Ngb-CO) are virtually identical. However, in contrast to Mb, human Ngb has an intramolecular disulfide bond that affects its oxygen affinity and protein stability. By using 2D-IR vibrational echo spectroscopy, we investigated the equilibrium protein dynamics of Ngb-CO by observing the CO spectral diffusion (time dependence of the 2D-IR line shapes) with and without the disulfide bond. Despite the similarity of the linear FTIR spectra of Ngb-CO with and without the disulfide bond, 2D-IR measurements reveal that the equilibrium sampling of different protein configurations is accelerated by disruption of the disulfide bond. The observations indicate that the intramolecular disulfide bond in Ngb acts as an inhibitor of fast protein dynamics even though eliminating it does not produce significant conformational change in the protein's structure.</P>
Thielges, Megan C.,Axup, Jun Y.,Wong, Daryl,Lee, Hyun Soo,Chung, Jean K.,Schultz, Peter G.,Fayer, Michael D. American Chemical Society 2011 The journal of physical chemistry. B, Condensed ma Vol.115 No.38
<P>Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform infrared (FT-IR) and 2D IR spectroscopic experiments on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chemical nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The experiments demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jpcbfk/2011/jpcbfk.2011.115.issue-38/jp206986v/production/images/medium/jp-2011-06986v_0004.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/jp206986v'>ACS Electronic Supporting Info</A></P>