Exploring the Molecular Origins of Protein Dynamics in the Active Site of Human Carbonic Anhydrase II

Hill, Sarah; Bandaria, Jigar N.; Fox, Michelle; Vanderah, Elizabeth; Kohen, Amnon; Cheatum, Christopher M.

doi:10.1021/jp901321m

Cited by 28 publications

(38 citation statements)

References 66 publications

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“…A close experimental approximation to this ideal is to study the dynamics of an enzyme in a complex with a transition-state-analog inhibitor. There have been several infrared spectroscopic studies of enzyme-ligand interaction dynamics with substrate-analog complexes that mimic the ground state (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). These studies use infrared echo and 2D IR spectroscopies to measure the frequencyfrequency time correlation function (FFCF) that reveals the time .…”

Section: D Ir Spectroscopy | Enzyme Dynamicsmentioning

confidence: 99%

Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase

Bandaria

Dutta

Nydegger

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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118

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The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzymecatalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using twodimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (N − 3 ). We observe that the frequency-frequency time correlation functions (FFCF) for the ternary complexes with NAD þ and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window. 2D IR spectroscopy | enzyme dynamicsT he functional role of protein motions at the femtosecond to picosecond time scale is a hotly debated topic in enzymology. Although such a role could be general in nature, many experimental (1-10) and theoretical (11-23) studies of enzymecatalyzed hydrogen transfers have invoked protein motions at this time scale to explain anomalous kinetic isotope effects (KIE) and their temperature dependence. These studies result in the development of theoretical models, often referred to as Marcus-like models, in which the environmental reorganization that precedes the hydrogen-tunneling event has evolved to optimize the conformation of the transition state for tunneling (1,7,24). Fig. 1 illustrates the physical picture underlying such models. Heavy atom motions along the reorganization coordinate carry the system to a point where the donor and acceptor wells in the double-well hydrogen atom potential are degenerate and tunneling can proceed. At this position, the donor-acceptor distance and its fluctuations determine the tunneling probability. Mathematically, the rate constant for hydrogen transfer in these models is given by expressions of the form where C is the fraction of reactive complexes, the first exponential, in analogy with the Marcus theory for electron transfer, reflects the reorganization of the heavy atoms that modulates the relative energies of the reactants and the products to minimize the energy defect between the zero-point levels of the donor and acceptor wells. ΔG°is the driving force for the reaction, and λ is the reorganization energy. The second exponential gives the overlap between the donor and acceptor wave functions as a function of the donor-acceptor distan...

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Section: D Ir Spectroscopy | Enzyme Dynamicsmentioning

confidence: 99%

Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase

Bandaria

Dutta

Nydegger

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

118

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“…40,43,45,48–51 The CO provides a single vibrational label with a strong IR transition located in specific, well-defined position. Similarly, studies have employed Az ion-ligands as IR probes, for example, bound to carbonic anhydrase, 14 hemoglobin, 14,19 and formate dehydrogenase. 12 …”

Section: Introductionmentioning

confidence: 99%

Two-Dimensional IR Spectroscopy of Protein Dynamics Using Two Vibrational Labels: A Site-Specific Genetically Encoded Unnatural Amino Acid and an Active Site Ligand

et al. 2011

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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.

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“…For example, the pseudohalide anions have been used to report both solvation dynamics [24][25][26][27][28][29] and protein dynamics. [30][31][32] These anions are nearly ideal spectroscopic probes because they exhibit strong transition moments, and their vibrational transitions, which are located between 2000 and 2200 cm −1 , are well separated from the spectral regions in which most organic molecules absorb, and these anions exhibit sharp absorption features and are sensitive to the fluctuations of their surroundings.…”

Section: Introductionmentioning

confidence: 99%

Two-dimensional infrared study of 3-azidopyridine as a potential spectroscopic reporter of protonation state

Nydegger

Dutta

Cheatum

2010

The Journal of Chemical Physics

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The lack of general spectroscopic probes that can be used in a range of systems to probe kinetics and dynamics is a major obstacle to the widespread application of two-dimensional infrared (2D IR) spectroscopy. We have studied 3-azidopyridine to characterize its potential as a probe of the protonation state of the pyridine ring. We find that the azido-stretching vibration is split by accidental Fermi resonance interactions with one or more overtones and combination states. Using 2D IR spectroscopy, we determine the state structure of the resulting eigenstates for complexes of 3-azidopyridine with formic acid and trifluoroacetic acid in which the pyridine ring is unprotonated and protonated, respectively. Based on the measurements, we develop a two-oscillator depurturbation model to determine the energies and couplings of the zeroth-order azido-stretching state and the perturbing dark state that couples to it. Based on these results, we conclude that the azido-stretching vibration is, in fact, sensitive to the protonation state of the pyridine shifting up in frequency by 8 cm(-1) in the complex with trifluoroacetic acid relative to the formic acid complex. These results suggest that, although 3-azidopyridine is not suitable as a spectroscopic probe, the approach of employing an organic azide as a remote probe of protonation state holds significant promise.

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Exploring the Molecular Origins of Protein Dynamics in the Active Site of Human Carbonic Anhydrase II

Cited by 28 publications

References 66 publications

Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase

Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase

Two-Dimensional IR Spectroscopy of Protein Dynamics Using Two Vibrational Labels: A Site-Specific Genetically Encoded Unnatural Amino Acid and an Active Site Ligand

Two-dimensional infrared study of 3-azidopyridine as a potential spectroscopic reporter of protonation state

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