Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins. It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown. Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions. To bind exogenous ligands like O2 and CO, the His E7 endogenous ligand is displaced from the sixth coordination. By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3-353 K). Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO. Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers. The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K. Flash photolysis near physiological temperature (275-353 K) exhibits four sequential kinetic features: (i) geminate rebinding (t < 1 s); (ii) extremely fast bimolecular exogenous ligand binding (10 s < t < 1 ms) with a nontrivial temperature dependence; (iii) endogenous ligand binding (100 s < t < 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (iv) displacement of the endogenous by the exogenous ligand (10 ms < t < 10 ks). All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales.G lobins are proteins that bind dioxygen and other small ligands at the central iron of a heme prosthetic group embedded in a highly conserved ␣-helical ''globin'' fold. Hemoglobin (Hb) and myoglobin (Mb) are the most prominent members of this protein family (1). Interrelations among structure, dynamics, and function in globins have been investigated in great detail, most likely more thoroughly than for any other protein family. Mb especially has, for a long time, served as a paradigm in the biological physics of proteins (2, 3).Recently, two new members have joined the globin family, cytoglobin (4) and neuroglobin (Ngb) (5). The latter consists of a single chain of 151 aa. Ngb, which occurs in neurons, has less than 25% sequence identity with Mb or Hb but nevertheless displays all key determinants of the globin fold (6). It has moderate oxygen affinity and seems most closely related to the globin found in the glial cells of the annelid Aphrodite (7). The discovery of a six-coordinate heme in the deoxy form of Ngb (5), with the His E7 side chain occupying the sixth coordination, came somewhat as a surprise because it was believed for a long time that a pentacoordinate heme iron, with a vacant ligandbinding site, was a common characteristic of globins. In recent years, however, hexacoordinate globins also have been isolated from bacteria, unicellular eukaryotes, and plants [nonsymbiotic Hbs, nsHbs (8,9), and truncated Hbs, trHbs (10)]. In these proteins, the exogenous ligand has to compete with an intramolecular side chain for ...

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4 -11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His 64 and Lys 67 . His 64 interferes with exogenous ligand access to the heme iron. Lys 67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.

We have studied the temperature dependence of the IR stretch bands of carbon monoxide (CO) in the Xe 4 internal cavity of myoglobin mutant L29W-S108L at cryogenic temperatures. Pronounced changes of band areas and positions were analyzed quantitatively by using a simple dynamic model in which CO rotation in the cavity is constrained by a static potential. The librational dynamics of the CO causes a decrease of the total spectral area. A strong local electric field splits the CO stretch absorption into a doublet, indicating that CO can assume opposite orientations in the cavity. With increasing temperature, the two peaks approach each other, because the average angle of the CO with respect to the electric field increases. A combined classical and quantum-mechanical analysis precisely reproduces the observed temperature dependencies of both spectral area and peak shifts. It yields the height of the energy barrier between the two wells associated with opposite CO orientations, V 0 Ϸ 2 kJ͞mol, and the frequency of oscillation within a well, Ϸ 25 cm ؊1 . The electric field in the protein cavity was estimated as 10 MV͞cm.E ven the simplest biological reactions exhibit a stunning level of complexity when studied in detail (1). The traditional chemical description in terms of transitions between a few discrete molecular species can only be a coarse approximation, because biological macromolecules are exceedingly complex physical systems that can assume a multitude of conformations (conformational substates) with markedly different structural and kinetic properties (2).Ligand binding in myoglobin (Mb), a small heme protein in mammalian muscle, has served for a long time as a biological model reaction. Pioneering low-temperature flash photolysis studies of CO and O 2 binding to Mb by Frauenfelder and coworkers (3) in the 70s gave clear evidence of protein structural heterogeneity as well as multiple intermediate states along the reaction pathway. Only in recent years have structural details of these intermediates become available. Fig. 1 shows a sketch of the free-energy surface governing ligand binding and a model of the active site of carboxymyoglobin (MbCO) (mutant L29W). After photodissociation, the CO moves from the bound-state location A to docking site B on top of the heme group. This primary photoproduct has been characterized by x-ray cryocrystallography of photolyzed MbCO crystals at 20-40 K (4-6). Fourier transform IR (FTIR) spectroscopy shows two distinct stretch bands for CO in state B (7, 8) that originate from two opposite orientations of the CO dipole with respect to an internal electric field. Recent femtosecond IR experiments (8) and time-resolved x-ray structure determinations (9) at room temperature have confirmed the relevance of docking site B in physiological ligand binding.Sites A and B are not the only internal locations available to ligands. Cryocrystallographic studies (10-12) have shown that, under proper illumination conditions, ligands can be chased into more remote, secondary docking sites C...

We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, A(I) and A(II), with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, A., et al. (2000) Nature 404, 205-208]. The detailed knowledge of the interplay between protein structure, protein dynamics, and ligand migration at cryogenic temperatures allowed us to develop a dynamic model that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.

An approach to automatically analyze and use the knowledge contained in electronic laboratory notebooks (ELNs) has been developed. Reactions were reduced to their reactive center and converted to a string representation (SMIRKS) which formed the basis for reaction classification and in silico (retro-)synthesis. Of the SMIRKS that occurred at least five times, 98% successfully regenerated the original product. The extracted reaction rules (SMIRKS) and corresponding reactants span a virtual chemical space which showed a strong dependence on the size of the reactive center. Whereas relatively few robust reaction types were sufficient to describe a large part of all reactions, considerably more reaction rules were necessary to cover all reactions in the ELN. Furthermore, reaction sequences were extracted to identify frequent combinations and diversifying reaction steps. Based on the extracted knowledge a (retro-)synthesis tool was built allowing for de novo design of compounds which have a high chance of being synthetically accessible. In an example application of the de novo design tool, various feasible retrosynthetic routes to the query molecule were obtained. Reaction based enumeration along the top ranked route yielded a library of 29 920 compounds with diverse properties, 99.9% of which are novel in the sense that they are unknown to the public domain.

To determine the magnitude and direction of the internal electric field in the Xe4 cavity of myoglobin mutant L29W-S108L, we have studied the vibrational Stark effect of carbon monoxide (CO) using infrared spectroscopy at cryogenic temperatures. CO was photodissociated from the heme iron and deposited selectively in Xe4. Its infrared spectrum exhibits Stark splitting into two bands associated with CO in opposite orientations. Two different photoproduct states can be distinguished, C' and C'', with markedly different properties. For C', characteristic temperature-dependent changes of the area, shift, and width were analyzed, based on a dynamic model in which the CO performs fast librations within a double-well model potential. For the barrier between the wells, a height of approximately 1.8 kJ/mol was obtained, in which the CO performs oscillations at an angular frequency of approximately 25 cm(-1). The magnitude of the electric field in the C' conformation was determined as 11.1 MV/cm; it is tilted by an angle of 29 degrees to the symmetry axis of the potential. Above 140 K, a protein relaxation leads to a significantly altered photoproduct, C'', with a smaller Stark splitting and a more confining potential (barrier >4 kJ/mol) governing the CO librations.

Recent years have seen a tremendous progress in the elucidation of experimental structural information for G-protein coupled receptors (GPCRs). Although for the vast majority of pharmaceutically relevant GPCRs structural information is still accessible only by homology models the steadily increasing amount of structural information fosters the application of structure-based drug design tools for this important class of drug targets. In this article we focus on the application of molecular dynamics (MD) simulations in GPCR drug discovery programs. Typical application scenarios of MD simulations and their scope and limitations will be described on the basis of two selected case studies, namely the binding of small molecule antagonists to the human CC chemokine receptor 3 (CCR3) and a detailed investigation of the interplay between receptor dynamics and solvation for the binding of small molecules to the human muscarinic acetylcholine receptor 3 (hM3R).