Conformational Dynamics and Stability of HP35 Studied with 2D IR Vibrational Echoes

Two-dimensional infrared (2D IR) vibrational echo spectroscopy was used to measure the fast dynamics of two variants of chicken villin headpiece 35 (HP35). The CN of cyanophenylalanine residues inserted in the hydrophobic core were used as a vibrational probe. Experiments were performed on both singly (HP35-P) and doubly CN-labeled peptide (HP35-P₂) within the wild-type sequence, as well as on HP-35 containing a singly labeled cyanophenylalanine and two norleucine mutations (HP35-P NleNle). There is a remarkable similarity between the dynamics measured in singly and doubly CN-labeled HP35, demonstrating that the presence of an additional CN vibrational probe does not significantly alter the dynamics of the small peptide. The substitution of two lysine residues by norleucines markedly improves the stability of HP35 by replacing charged with nonpolar residues, stabilizing the hydrophobic core. The results of the 2D IR experiments reveal that the dynamics of HP35-P are significantly faster than those of HP35-P NleNle. These observations suggest that the slower structural fluctuations in the hydrophobic core, indicating a more tightly structured core, may be an important contributing factor to HP35-P NleNle’s increased stability

Conformational Landscape and the Selectivity of Cytochrome P450cam

Conformational heterogeneity and dynamics likely contribute to the remarkable activity of enzymes but are challenging to characterize experimentally. These features are of particular interest within the cytochrome P450 class of monooxygenases, which are of great academic, medicinal, and biotechnological interest as they recognize a broad range of substrates, such as various lipids, steroid precursors, and xenobiotics, including therapeutics. Here, we use linear and 2D IR spectroscopy to characterize the prototypical P450, cytochrome P450cam, bound to three different substrates, camphor, norcamphor, or thiocamphor, which are hydroxylated with high, low, and intermediate regioselectivity, respectively. The data suggest that specific interactions with the substrate drive the population of two different conformations, one that is associated with high regioselectivity and another associated with lower regioselectivity. Although Y96 mediates a hydrogen bond thought necessary to orient the substrate for high regioselectivity, the population and dynamics of the conformational states are largely unaltered by the Y96F mutation. This study suggests that knowledge of the conformational landscape is central to understanding P450 activity, which has important practical ramifications for the design of therapeutics with optimized pharmacokinetics, and the manipulation of P450s, and possibly other enzymes, for biotechnological applications

Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy

Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated <i>p</i>-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function

Fast Dynamics of HP35 for Folded and Urea-Unfolded Conditions

The changes in fast dynamics of HP35 with a double CN vibrational dynamics label (HP35-P₂) as a function of the extent of denaturation by urea were investigated with two-dimensional infrared (2D IR) vibrational echo spectroscopy. Cyanophenylalanine (PheCN) replaces the native phenylalanine at two residues in the hydrophobic core of HP35, providing vibrational probes. NMR data show that HP35-P₂ maintains the native folded structure similar to wild type and that both PheCN residues share essentially the same environment within the peptide. A series of time-dependent 2D IR vibrational echo spectra were obtained for the folded peptide and the increasingly unfolded peptide. Analysis of the time dependence of the 2D spectra yields the system’s spectral diffusion, which is caused by the sampling of accessible structures of the peptide under thermal equilibrium conditions. The structural dynamics become faster as the degree of unfolding is increased

Carbon−Deuterium Bonds as Site-Specific and Nonperturbative Probes for Time-Resolved Studies of Protein Dynamics and Folding

Carbon−deuterium (C−D) bonds are nonperturbative spectroscopic probes that absorb in a region of the IR spectrum that is free of other protein absorptions. We explore the use of these probes under time-resolved conditions to follow the unfolding of cytochrome <i>c</i> from a photostationary state that accumulates after CO is photodissociated from the protein’s heme prosthetic group. Our results clearly show that C−D bonds are well-suited to characterize protein folding and dynamics

Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy

The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon–deuterium (C–D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn­(II) or Co­(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (<i>d</i>₃-<i>methyl</i>)­Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn–Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) <i>f</i>. The spectral changes suggest a slightly stronger Cu–S­(Met97) interaction in the complex with cyt <i>f</i> that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C–D probes

Engineering a Conformationally Switchable Artificial Metalloprotein

Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co­(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3–, H3C–, and iPr–). We used UV–vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co–S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co­(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co–S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation