Biomedical subjects

Karin Nienhaus

Publications and source records attributed to Karin Nienhaus.

At least 19 recordsLinked to original sources

PubMed2006

Exploring chromophore--protein interactions in fluorescent protein cmFP512 from Cerianthus membranaceus: X-ray structure analysis and optical spectroscopy.

Autofluorescent proteins of the GFP family all share the same three-dimensional beta-can fold; yet they exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic characterization of the green fluorescent protein cmFP512 from Cerianthus membranaceus, a nonbioluminescent, azooxanthellate cnidarian, which has only approximately 22% sequence identity with Aequorea victoria GFP. The X-ray structure, obtained by molecular replacement at a resolution of 1. 35 A, shows the chromophore, formed from the tripeptide Gln-Tyr-Gly, in a hydrogen-bonded cage in the center of an 11-stranded beta-barrel, tightly restrained by adjacent residues and structural water molecules. It exists in a neutral (A) and an anionic (B) species, with absorption/emission maxima at 392/460 (pH 5) and 503/512 nm (pH 7). Their fractional populations and peak positions depend sensitively on pH, reflecting protonation of groups adjacent to the chromophore. The pH dependence of the spectra is explained by a protonation mechanism involving a hydrogen-bonded cluster of charged/polar groups. Cryospectroscopy at 12 K was also performed to analyze the vibronic coupling of the electronic transitions.

Animals↗

PubMed2006

Chromophore-protein interactions in the anthozoan green fluorescent protein asFP499.

Despite their similar fold topologies, anthozoan fluorescent proteins (FPs) can exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic investigation of the green FP asFP499 from the sea anemone Anemonia sulcata var. rufescens to explore the effects of the protein environment on the chromophore. The optical absorption and fluorescence spectra reveal two discrete species populated in significant proportions over a wide pH range. Moreover, multiple protonation reactions are evident from the observed pH-dependent spectral changes. The x-ray structure of asFP499, determined by molecular replacement at a resolution of 1.85 A, shows the typical beta-barrel fold of the green FP from Aequorea victoria (avGFP). In its center, the chromophore, formed from the tripeptide Gln(63)-Tyr(64)-Gly(65), is tightly held by multiple hydrogen bonds in a polar cage that is structurally quite dissimilar to that of avGFP. The x-ray structure provides interesting clues as to how the spectroscopic properties are fine tuned by the chromophore environment.

Animals↗

PubMed2006

Spectroscopic study of substrate binding to the carbonmonoxy form of dehaloperoxidase from Amphitrite ornata.

Dehaloperoxidase (DHP) is a globular heme enzyme found in the marine worm Amphitrite ornata that can catalyze the dehalogenation of halophenols to the corresponding quinones by using hydrogen peroxide as a cosubstrate. Its three-dimensional fold is surprisingly similar to that of the oxygen storage protein myoglobin (Mb). A key structural feature common to both DHP and Mb is the existence of multiple conformations of the distal histidine. In DHP, the conformational flexibility may be involved in promotion of substrate and cosubstrate entry and exit. Here we have explored the dynamics of substrate binding in DHP using Fourier transform infrared spectroscopy and flash photolysis. A number of discrete conformations at the active site were identified from the appearance of multiple CO absorbance bands in the infrared region of the spectrum. Upon photolysis at cryogenic temperatures, the CO molecules are trapped at docking sites within the protein matrix, as inferred from the appearance of several photoproduct bands characteristic of each site. Substrate binding stabilizes the protein by approximately 20 kJ/mol. The low yield of substrate-bound DHP at ambient temperature points toward a steric inhibition of substrate binding by carbon monoxide.

Animals↗

PubMed2005

Ligand migration pathway and protein dynamics in myoglobin: a time-resolved crystallographic study on L29W MbCO.

By using time-resolved x-ray crystallography at room temperature, structural relaxations and ligand migration were examined in myoglobin (Mb) mutant L29W from nanoseconds to seconds after photodissociation of carbon monoxide (CO) from the heme iron by nanosecond laser pulses. The data were analyzed in terms of transient kinetics by fitting trial functions to integrated difference electron density values obtained from select structural moieties, thus allowing a quantitative description of the processes involved. The observed relaxations are linked to other investigations on protein dynamics. At the earliest times, the heme has already completely relaxed into its domed deoxy structure, and there is no photo-dissociated CO visible at the primary docking site. Initial relaxations of larger globin moieties are completed within several hundred nanoseconds. They influence the concomitant migration of photo-dissociated CO to the Xe1 site, where it appears at approximately 300 ns and leaves again at approximately 1.5 ms. The extremely long residence time in Xe1 as compared with wild-type MbCO implies that, in the latter protein, the CO exits the protein from Xe1 predominantly via the distal pocket. A well-defined deligated state is populated between approximately 2 micros and approximately 1 ms; its structure is very similar to the equilibrium deoxy structure. Between 1.5 and 20 ms, no CO is visible in the protein interior; it is either distributed among many sites within the protein or has escaped to the solvent. Finally, recombination at the heme iron occurs after >20 ms.

Amino Acid Substitution↗

PubMed2005

Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP.

Genetically encoded fusion constructs derived from fluorescent proteins (FPs) can be designed to report on a multitude of events and signals in cells, tissues, and entire organs without interfering with the complex machinery of life. EosFP is a novel FP from the scleractinian coral Lobophyllia hemprichii that switches its fluorescence emission from green (516 nm) to red (581 nm) upon irradiation with approximately 400-nm light. This property enables localized tagging of proteins and thus provides a valuable tool for tracking protein movements within live cells. Here, we present the x-ray structures of the green and red forms of WT EosFP. They reveal that formation of the red chromophore is associated with cleavage of the peptide backbone, with surprisingly little change elsewhere in the structure, and provide insights into the mechanism that generates this interesting posttranslational polypeptide modification.

Animals↗

PubMed2005

Neuroglobin, nitric oxide, and oxygen: functional pathways and conformational changes.

Neuroglobin (Ngb) is a globin expressed in the nervous system of humans and other organisms that is involved in the protection of the brain from ischemic damage. Despite considerable interest, however, the in vivo function of Ngb is still a conundrum. In this paper we report a number of kinetic experiments with O2 and NO that we have interpreted on the basis of the 3D structure of Ngb, now available for human and murine metNgb and murine NgbCO. The reaction of reduced deoxyNgb with O2 and NO is slow (t(1/2) approximately 2 s) and ligand concentration-independent, because exogenous ligand binding can only occur upon dissociation of the distal His-64, which is coordinated to the ferrous heme iron. By contrast, NgbO2 reacts very rapidly with NO, yielding metNgb and NO3- by means of a heme-bound peroxynitrite intermediate. Steady-state amperometric experiments show that Ngb is devoid of O2 reductase and NO reductase activities. To achieve this result, we have set up a protocol for efficient reduction of metNgb using a mixture of FMN and NADH under bright illumination. The results are discussed with reference to a global scheme inspired by the 3D structures of metNgb and NgbCO. Based on the ligand-linked conformational changes discovered by crystallography, the pathways of the reactions with O2 and NO provide a framework that may account for the involvement of Ngb in controlling the activation of a protective signaling mechanism.

Animals↗

PubMed2005

Ligand migration and protein fluctuations in myoglobin mutant L29W.

We have determined eight X-ray structures of myoglobin mutant L29W at various experimental conditions. In addition, infrared spectroscopic experiments are presented, which are discussed in the light of the X-ray structures. Two distinct conformations of the CO-ligated protein were identified, giving rise to two stretching bands of heme-bound CO. If L29W MbCO crystals are illuminated around 180 K, a deoxy species is formed. The CO molecules migrate to the proximal side of the heme and remain trapped in the so-called Xe1 cavity upon temperature decrease to 105 K. The structure of this photoproduct is almost identical to the equilibrium high-temperature deoxy Mb structure. If the temperature is cycled to increasingly higher values, CO recombination is observed. Three intermediate structures have been determined during the rebinding process. Efficient recombination occurs only above 180 K, the characteristic temperature for the onset of protein dynamics. Rebinding is remarkably slow because bulky residues His64 and Trp29 block important migration pathways of the CO molecule.

Amino Acid Substitution↗

PubMed2005

Restricted rotational motion of CO in a protein internal cavity: evidence for nonseparating correlation functions from IR pump-probe spectroscopy.

The strongly restricted orientational motion of CO molecules trapped in the Xe4 internal cavity of myoglobin mutant L29W-S108L is investigated by polarization-dependent mid-infrared pump-probe spectroscopy at cryogenic temperatures. Following an ultrafast initial decay, the signal anisotropy reaches an asymptotic value that is significantly larger than the prediction from the well-known relation [see text], based on previously established potential parameters. This discrepancy is explained by showing that the full four-point correlation function describing third-order spectroscopy [see text] does not factorize in systems where its fast decay is dominated by restricted reorientation of the transition dipole moments.

Animals↗

PubMed2005

The origin of stark splitting in the initial photoproduct state of MbCO.

Ligand migration and binding in heme proteins have been measured by X-ray diffraction and time-resolved spectroscopy of photoproduct intermediates. In myoglobin (Mb), internal cavities serve as docking sites for carbon monoxide (CO) ligands. In these sites, the CO ligands display characteristic infrared (IR) stretching bands due to interactions with the local electrical field. In the primary docking site, a CO can reside in two opposite orientations, characterized by a doublet of infrared bands, B1 at approximately 2130 and B2 at approximately 2120 cm-1. To assign these bands to the specific orientations, we have reexamined the effects of mutating His64 and Val68 on the infrared stretching bands associated with the B1 and B2 photoproduct states. Wild-type, H64L, V68F, and H64L-V68F MbCO were selected for experimental and theoretical analyses. Fourier transform infrared (FTIR) spectroscopy and density functional theory (DFT) calculations were used to interpret the effects of the electrostatic environment on the B state bands. The imidazole side chain of His64 appears to be the primary cause of the observed Stark splitting. The high-frequency B1 band is assigned to the CO orientation in which the carbon (white atom) is directed toward the heme iron and the Nepsilon-H proton of His64. At low temperatures, CO molecules in the opposite orientational conformer, B2 with the O atom (red) toward His64, first rotate by 180 degrees into the more stable B1 state and then rebind.

Carbon Monoxide↗

PubMed2005

Probing heme protein-ligand interactions by UV/visible absorption spectroscopy.

Ultraviolet/visible (UV/vis) absorption spectroscopy is a powerful tool for steady-state and time-resolved studies of protein-ligand interactions. Prosthetic groups in proteins frequently have strong electronic absorbance bands that depend on the oxidation, ligation, and conformation states of the chromophores. They are also sensitive to conformational changes of the polypeptide chain into which they are embedded. Steady-state absorption spectroscopy provides information on ligand binding equilibria, from which the Gibbs free energy differences between the ligated and unligated states can be computed. Time-resolved absorption spectroscopy allows one to detect short-lived intermediate states that may not get populated significantly under equilibrium conditions, but may nevertheless be of crucial importance for biological function. Moreover, the energy barriers that have to be surmounted in the reaction can be determined. In this chapter, we present a number of typical applications of steady-state and ns time-resolved UV/vis absorption spectroscopy in the study of ligand binding to the central iron in heme proteins.

Animals↗

PubMed2004

Probing electric fields in protein cavities by using the vibrational stark effect of carbon monoxide.

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.

Animals↗

PubMed2004

The structure of carbonmonoxy neuroglobin reveals a heme-sliding mechanism for control of ligand affinity.

Neuroglobin (Ngb), a globular heme protein expressed in the brain of vertebrates, binds oxygen reversibly, with an affinity comparable to myoglobin (Mb). Despite low sequence identity, the overall 3D fold of Ngb and Mb is very similar. Unlike in Mb, in Ngb the sixth coordination position of the heme iron is occupied by the distal histidine, in the absence of an exogenous ligand. Endogenous ligation has been proposed as a unique mechanism for affinity regulation and ligand discrimination in heme proteins. This peculiarity might be related to the still-unknown physiological function of Ngb. Here, we present the x-ray structure of CO-bound ferrous murine Ngb at 1.7 A and a comparison with the 1.5-A structure of ferric bis-histidine Ngb. We have also used Fourier transform IR spectroscopy of WT and mutant CO-ligated Ngb to examine structural heterogeneity in the active site. Upon CO binding, the distal histidine retains (by and large) its position, whereas the heme group slides deeper into a preformed crevice, thereby reshaping the large cavity ( approximately 290 A(3)) connecting the distal and proximal heme sides with the bulk. The heme relocation is accompanied by a significant decrease of structural disorder, especially of the EF loop, which may be the signal whereby Ngb communicates hypoxic conditions. This unexpected structural change unveils a heme-sliding mechanism of affinity control that may be of significance to understanding Ngb's role in the pathophysiology of the brain.

Amino Acid Sequence↗

PubMed2004

Structural dynamics controls nitric oxide affinity in nitrophorin 4.

Nitrophorin 4 (NP4) is one of seven nitric oxide (NO) transporting proteins in the blood-sucking insect Rhodnius prolixus. In its physiological function, NO binds to a ferric iron centered in a highly ruffled heme plane. Carbon monoxide (CO) also binds after reduction of the heme iron. Here we have used Fourier transform infrared spectroscopy at cryogenic temperatures to study CO and NO binding and migration in NP4, complemented by x-ray cryo-crystallography on xenon-containing NP4 crystals to identify cavities that may serve as ligand docking sites. Multiple infrared stretching bands of the heme-bound ligands indicate different active site conformations with varying degrees of hydrophobicity. Narrow infrared stretching bands are observed for photodissociated CO and NO; temperature-derivative spectroscopy shows that these bands are associated with ligand docking sites close to the extremely reactive heme iron. No rebinding from distinct secondary sites was detected, although two xenon binding cavities were observed in the x-ray structure. Photolysis studies at approximately 200 K show efficient NO photoproduct formation in the more hydrophilic, open NP4 conformation. This result suggests that ligand escape is facilitated in this conformation, and blockage of the active site by water hinders immediate reassociation of NO to the ferric iron. In the closed, low-pH conformation, ligand escape from the active site of NP4 is prevented by an extremely reactive heme iron and the absence of secondary ligand docking sites.

Animals↗

PubMed2004

The structure of murine neuroglobin: Novel pathways for ligand migration and binding.

Neuroglobin, a recently discovered globin predominantly expressed in neuronal tissue of vertebrates, binds small, gaseous ligands at the sixth coordination position of the heme iron. In the absence of an exogenous ligand, the distal histidine (His64) binds to the heme iron in the ferrous and ferric states. The crystal structure of murine ferric (met) neuroglobin at 1.5 A reveals interesting features relevant to the ligand binding mechanism. Only weak selectivity is observed for the two possible heme orientations, the occupancy ratio being 70:30. Two small internal cavities are present on the heme distal side, which enable the His64(E7) side chain to move out of the way upon exogenous ligand binding. Moreover, a third, huge cavity (volume approximately 290 A3) connecting both sides of the heme, is open towards the exterior and provides a potential passageway for ligands. The CD and EF corners exhibit substantial flexibility, which may assist ligands in entering the protein and accessing the active site. Based on this high-resolution structure, further structure-function studies can be planned to elucidate the role of neuroglobin in physiological responses to hypoxia.

Animals↗

PubMed2004

Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin.

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 = 200-600 s(-1)) and moderate O2 affinity (KO2) approximately 1 microm(-1)) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 degrees C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 --> Val CerHbO2 at 1.70 A resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 A). The dramatic functional and spectral effects of the Thr-E11 --> Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm(-1) decrease in nuCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.

Animals↗

PubMed2004

Structural dynamics of myoglobin: an infrared kinetic study of ligand migration in mutants YQR and YQRF.

Recombination of carbon monoxide to myoglobin mutants YQR and YQRF was studied using transient infrared absorption spectroscopy and Fourier transform infrared-temperature derivative spectroscopy (FTIR-TDS). Photoproduct states B, C', C" and D associated with ligands residing in different protein cavities have been identified. After photolysis, ligands migrate to primary docking site B and subsequently rebind or escape to a secondary site (C) within the Xe4 cavity. For YQR, a global analysis of the isothermal rebinding kinetics below 160 K and the TDS data reveal a correlation between the enthalpy barriers governing the two processes. Above 120 K, a protein conformational change in both YQR and YQRF converts photoproduct C' into C" with markedly slowed kinetics. Above approximately 180 K, ligands migrate to the proximal Xe1 site (D) and also exit into the solvent, from where they rebind in a bimolecular reaction.

Carbon Monoxide↗

PubMed2004

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding.

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 His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 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.

Animals↗

PubMed2004

The effect of protein internal cavities on ligand migration and binding in myoglobin.

Using Fourier Transform Infrared Spectroscopy at cryogenic temperatures, we have studied carbon monoxide (CO) migration in the interior of sperm whale myoglobin and binding to teh heme iron. The effect of protein internal cavities was examined by comparing the wild-type protein with mutants in which the cavities were blocked by bulky amino acid sidechains. After photodissociation at 3 K, CO ligands reside in the primary docking site (B) from where they can migrate to other sites (C, D) that were identified as the Xe4 and Xe1 cavities. These studies were complemented by flash photolysis experiments at room temperature, which revealed that the protein cavities enable efficient excape of ligands from the protein after dissociation from the heme iron.

Animals↗