Protein-ligand dynamics: A 96 picosecond simulation of a myoglobin-xenon complex

doi:10.1016/0022-2836(88)90389-0

Journal of Molecular Biology

Volume 199, Issue 1, 5 January 1988, Pages 195-211

https://doi.org/10.1016/0022-2836(88)90389-0 Get rights and content

Abstract

A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal cavities is examined to study the effect of protein motions on ligand motion and internal cavity fluctuations. Average structural and energetic properties indicate that the simulation is well behaved. The average protein volume is similar to the volume of the X-ray model and the main-chain atom root-mean-square deviation between the X-ray model and the average dynamical structure is 1.25 Å. The protein volume oscillates 3 to 4% around the volume of the X-ray structure. These fluctuations lead to changes in the internal free volume and in the size, shape and location of atom-sized cavity features. Transient cavities produced in the simulation have a crucial role in the movement of two of the ligands. One of the ligands escapes to the protein surface, whilst a second ligand travels through the protein interior. Complex gating processes involving several protein residues are responsible for producing the necessary pores through which the ligand passes between transient cavities or packing defects.

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Xenon–Protein Interactions: Characterization by X-Ray Crystallography and Hyper-CEST NMR
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Increasing the concentration of myoglobin in solution moves the chemical shift of the lone NMR peak either upfield or downfield depending on the oxidation state and spin state of the heme Fe (Rubin, Lee, Ruiz, Pines, & Wemmer, 2002; Tilton & Kuntz, 1982). In silico analysis of Xe–myoglobin interactions suggest that Xe exchange is controlled by complex gating processes in which structural fluctuations within the protein control the rate of Xe access to the buried interior binding cavity (Teeter, 2004; Tilton, Singh, Kuntz, & Kollman, 1988). Finally, although Xe binding minimally perturbs myoglobin structure, Xe (as well as nitrogen and cyclopropane) binding to myoglobin alters its function, specifically its binding affinity for carbon monoxide (Settle, 1973).
The physiological activity of xenon has long been recognized, though the exact nature of its interactions with biomolecules remains poorly understood. Xe is an inert noble gas, but can act as a general anesthetic, most likely by binding internal hydrophobic cavities within proteins. Understanding Xe–protein interactions, therefore, can provide crucial insight regarding the mechanism of Xe anesthesia and potentially other general anesthetic agents. Historically, Xe–protein interactions have been studied primarily through X-ray crystallography and nuclear magnetic resonance (NMR). In this chapter, we first describe our methods for preparing Xe derivatives of protein crystals and identifying Xe-binding sites. Second, we detail our procedure for ¹²⁹Xe hyper-CEST NMR spectroscopy, a versatile NMR technique well suited for characterizing the weak, transient nature of Xe–protein interactions.
An atomistic view on human hemoglobin carbon monoxide migration processes
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Most studies indicate that this is the most favorable and shortest pathway for ligands to reach or escape the heme (20). In addition, there is evidence for other paths on the distal side of the protein as well as passing by the experimentally observed xenon sites (23,24). Many years of intense research was necessary to unravel the putative network of pathways known nowadays in Mb (3,25–29).
A significant amount of work has been devoted to obtaining a detailed atomistic knowledge of the human hemoglobin mechanism. Despite this impressive research, to date, the ligand diffusion processes remain unclear and controversial. Using recently developed computational techniques, PELE, we are capable of addressing the ligand migration processes. First, the methodology was tested on myoglobin's CO migration, and the results were compared with the wealth of theoretical and experimental studies. Then, we explored both hemoglobin tense and relaxed states and identified the differences between the α-and β-subunits. Our results indicate that the proximal site, equivalent to the Xe1 cavity in myoglobin, is never visited. Furthermore, strategically positioned residues alter the diffusion processes within hemoglobin's subunits and suggest that multiple pathways exist, especially diversified in the α-globins. A significant dependency of the ligand dynamics on the tertiary structure is also observed.
Ligand dynamics in heme proteins observed by Fourier transform infrared-temperature derivative spectroscopy
2011, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
Presently, this study provides the strongest evidence that the “histidine gate” is the predominant ligand entry and exit pathway. In 1966, Perutz and Matthews [45] had proposed that the pathway for ligand entry and exit into mammalian Hbs and Mbs involved rotation of the distal histidine, HisE7, to form a short and direct channel between the heme pocket and solvent, an idea that has been discussed controversially ever since [31,32,44,46–48]. FTIR-TDS has been a key method to identify photoproduct states, to characterize their rebinding properties and to enhance their population for cryo-crystallography experiments in MbCO.
Fourier transform infrared (FTIR) spectroscopy is a powerful tool for the investigation of protein–ligand interactions in heme proteins. Nitric oxide and carbon monoxide are attractive physiologically relevant ligands because their bond stretching vibrations give rise to strong mid-infrared absorption bands that can be measured with exquisite sensitivity and precision using photolysis difference spectroscopy at cryogenic temperatures. These stretching bands are fine-tuned by electrostatic interactions with the environment and, therefore, ligands can be utilized as local probes of structure and dynamics. Bound to the heme iron, the ligand stretching bands are susceptible to changes in the iron–ligand bond and the electric field at the active site. Upon photolysis, the vibrational bands display changes due to ligand relocation to docking sites within the protein, rotational motions of the ligand in these sites and protein conformational changes. Photolysis difference spectra taken over a wide temperature range (3–300 K) using specific temperature protocols for sample photodissociation can provide detailed insights into both protein and ligand dynamics. Moreover, temperature-derivative spectroscopy (TDS) has proven to be a particularly powerful technique to study protein–ligand interactions. The FTIR-TDS technique has been extensively applied to studies of carbon monoxide binding to heme proteins, whereas measurements with nitric oxide are still scarce. Here we describe infrared cryo-spectroscopy and present a variety of applications to the study of protein–ligand interactions in heme proteins. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.
Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells
2011, Life Sciences
Citation Excerpt :
Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand-binding and substrate-binding pockets and into the pore of channel-like structures. Xenon, through weak van der Waals forces, can bind to pre-existing atomic-sized cavities in the interior of certain globular protein molecules (Schoenborn and Nobbs, 1966; Tilton et al. 1986; Tilton et al. 1988; Schiltz et al. 1995; Prange et al. 1998; Labella et al. 1999). Xenon demonstrates an exceptional promiscuity, even binding to tissue-plasminogen activator, which is a serine protease (David et al., 2010).
Xenon provides effective analgesia in several pain states at sub-anaesthetic doses. Our aim was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of transient receptor potential vanilloid type 1 (TRPV1), a receptor known to be involved in certain inflammatory pain conditions.
We studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. We also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin.
Xenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM–1 μM) by ~ 25% to ~ 50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~ 50%. Xenon (80%) reduced by ~ 40% the number of phosporylated ERK1/2-expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection.
Xenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation.
Ligand migration in the truncated hemoglobin-II from Mycobacterium tuberculosis: The role of G8 tryptophan
2009, Journal of Biological Chemistry
Resonance Raman studies show that the heme-bound CO in trHbO, a truncated-II hemoglobin from Mycobacterium tuberculosis, is exposed to an environment with a positive electrostatic potential. The mutation of Trp(G8), an absolutely conserved residue in group II and III truncated hemoglobins, to Phe introduces two new Fe–CO conformers, both of which exhibit reduced electrostatic potentials. Computer simulations reveal that the structural perturbation is a result of the increased flexibility of the Tyr(CD1) and Leu(E11) side chains due to the reduction of the size of the G8 residue. Laser flash photolysis studies show that the G8 mutation induces 1) the presence of two new geminate recombination phases, one with a rate faster than the time resolution of our instrument and the other with a rate 13-fold slower than that of the wild type protein, and 2) the reduction of the total geminate recombination yield from 86 to 62% and the increase in the bimolecular recombination rate by a factor of 530. Computer simulations uncover that the photodissociated ligand migrates between three distal temporary docking sites before it subsequently rebinds to the heme iron or ultimately escapes into the solvent via a hydrophobic tunnel. The calculated energy profiles associated with the ligand migration processes are in good agreement with the experimental observations. The results highlight the importance of the Trp(G8) in regulating ligand migration in trHbO, underscoring its pivotal role in the structural and functional properties of the group II and III truncated hemoglobins.
Structural and functional properties of a truncated hemoglobin from a food-borne pathogen Campylobacter jejuni
2007, Journal of Biological Chemistry
Campylobacter jejuni contains two hemoglobins, Cgb and Ctb. Cgb has been suggested to perform an NO detoxification reaction to protect the bacterium against NO attack. On the other hand, the physiological function of Ctb, a class III truncated hemoglobin, remains unclear. By using CO as a structural probe, resonance Raman data show that the distal heme pocket of Ctb exhibits a positive electrostatic potential. In addition, two ligand-related vibrational modes, ν_Fe-O₂ and ν_O-O, were identified in the oxy derivative, with frequencies at 542 and 1132 cm^-1, respectively, suggesting the presence of an intertwined H-bonding network surrounding the heme-bound ligand, which accounts for its unusually high oxygen affinity (222 μm^-1). Mutagenesis studies of various distal mutants suggest that the heme-bound dioxygen is stabilized by H-bonds donated from the Tyr(B10) and Trp(G8) residues, which are highly conserved in the class III truncated hemoglobins; furthermore, an additional H-bond donated from the His(E7) to the Tyr(B10) further regulates these H-bonding interactions by restricting the conformational freedom of the phenolic side chain of the Tyr(B10). Taken together, the data suggest that it is the intricate balance of the H-bonding interactions that determines the unique ligand binding properties of Ctb. The extremely high oxygen affinity of Ctb makes it unlikely to function as an oxygen transporter; on the other hand, the distal heme environment of Ctb is surprisingly similar to that of cytochrome c peroxidase, suggesting a role of Ctb in performing a peroxidase or P450-type of oxygen chemistry.

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^☆: This work was supported by the following grants: GM31497, GM29072 and F32-AM07339-03.

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Journal of Molecular Biology

Protein-ligand dynamics: A 96 picosecond simulation of a myoglobin-xenon complex☆

Abstract

J. Mol. Biol

Methods Enzymol

J. Mol. Biol

J. Mol. Biol

J. Mol. Biol

Biophys. J

J. Mol. Biol

J. Mol. Biol

J. Mol. Biol

Biochemistry

J. Chem. Phys

J. Chem. Soc. London Faraday Trans

Science

J. Appl. Crystallogr

Int. J. Pept. Protein Res

Biochemistry