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1.
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. 相似文献
2.
Relaxations and fluctuations in myoglobin 总被引:1,自引:0,他引:1
One major goal of biological physics is the discovery and understanding of the concepts and laws that govern biomolecules, in particular proteins. Since there exist at least 10(5) different proteins, the choice of a suitable prototype is necessary. Myoglobin (Mb) has for many years played the role of such a prototype. It appears to be simple enough so that many of its properties can be understood, yet it is complex enough to display many of the fascinating characteristics of biomolecules. One major achievement in the study of any protein would be the establishment of convincing connections among structure, kinetics, energy landscape, dynamics, and function. We believe that this goal has not yet been reached in any protein, but the present knowledge of Mb gives some hope that the end is near in this case. Here, we sketch some of the results that have been obtained in the past 50 or more years in the research on Mb, obtained by an army of investigators. 相似文献
3.
The angular dependencies of inelastic intensities of Rayleigh scattering of Moessbauer radiation were measured for myoglobin and lysozyme (in the hydration range h = 0.05-0.7). The data were fitted within the framework of model, when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative motions of segments. The best agreement with the experiment at h > 0.05 was obtained when individual motions of small groups together with the cooperative motions of alpha-helices and beta-sheets for lysozyme, and alpha-helices for myoglobin were considered. At further hydration (h = 0.45), mean-square displacements (x2) of both types of motions strongly increase with the increase in hydration degree, while the motions with a large correlation radius (not less than macromolecule radius) remain nearly the same as for h = 0.05. The results of the study of the radial distribution function deduced by Fourier-transform from the diffuse x-ray measurements together with RSMR data allow one to conclude that the water during protein hydration competes with the intramolecular hydrogen bonds, loosens the protein and increases the internal dynamics. Concurrently, water arranges the ordering of macromolecule, which takes the native structure at h = 0.4-0.7. The analysis of auto and cross-correlation functions of bending fluctuations of alpha-helices in the large domain of lysozyme performed by molecular dynamics allows one to come to the final conclusion that it is the difference in the structural organization of myoglobin and lysozyme and not the presence of SS-bonds in lysozyme macromolecule that is responsible for different structural fluctuations in these proteins. 相似文献
4.
Agmon N 《Biophysical journal》2004,87(3):1537-1543
Protein relaxation, ligand binding, and ligand migration into a hydrophobic cavity in myoglobin are unified by a bounded diffusion model which produces an accurate fit to complex ligand rebinding data over eight decades in time and a 160 K temperature range, in qualitative agreement with time-resolved x-ray crystallography. Protein relaxation operates in a cyclic manner to move the ligand away from the binding site. 相似文献
5.
Hara I Ueno T Ozaki Si Itoh S Lee K Ueyama N Watanabe Y 《The Journal of biological chemistry》2001,276(39):36067-36070
The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the (1)H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The (13)C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH(2) group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system. 相似文献
6.
7.
Myoglobin (Mb) is a model system for ligand binding and migration. The energy barriers (ΔG) for ligand migration in Mb have been studied in the past by experiment and theory and significant differences between different approaches were found. From experiment, it is known that Mb can assume a large number of conformational substates. In this work, these substates are investigated as a possible source of the differences in migration barriers. We show that the initial structure significantly affects the calculated ΔG for a particular transition and that fluctuations in barrier heights δΔG are of similar magnitude as the free energy barriers themselves. The sensitivity of ΔG to the initial structure is compared to other sources of errors. Different protein structures can affect the calculated ΔG by up to 4 kcal/mol, whereas differences between simple point charge models and more elaborate multipolar charge models for the CO-ligand are smaller by a factor of two. Analysis of the structural changes underlying the large effect of the conformational substate reveals the importance of coupling between protein and ligand motion for migration. 相似文献
8.
Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively conservative change of a surface residue can substantially perturb ligand and proton exchange dynamics in a manner that is not readily predicted from the static structures. 相似文献
9.
Margaret M Stratton Thomas A Cutler Jeung-Hoi Ha Stewart N Loh 《Protein science : a publication of the Protein Society》2010,19(8):1587-1594
All proteins undergo local structural fluctuations (LSFs) or breathing motions. These motions are likely to be important for function but are poorly understood. LSFs were initially defined by amide hydrogen exchange (HX) experiments as opening events, which expose a small number of backbone amides to 1H/2H exchange, but whose exchange rates are independent of denaturant concentration. Here, we use size-dependent thiol-disulfide exchange (SX) to characterize LSFs in single cysteine-containing variants of myoglobin (Mb). SX complements HX by providing information on motions that disrupt side chain packing interactions. Most importantly, probe reagents of different sizes and chemical properties can be used to characterize the size of structural opening events and the properties of the open state. We use thiosulfonate reagents (126–274 Da) to survey access to Cys residues, which are buried at specific helical packing interfaces in Mb. In each case, the free energy of opening increases linearly with the radius of gyration of the probe reagent. The slope and the intercept are interpreted to yield information on the size of the opening events that expose the buried thiol groups. The slope parameter varies by over 10-fold among Cys positions tested, suggesting that the sizes of breathing motions vary substantially throughout the protein. Our results provide insight to the longstanding question: how rigid or flexible are proteins in their native states? 相似文献
10.
The pathways for ligand entry and exit in myoglobin have now been well established by a wide variety of experimental results, including pico- to nano- to microsecond transient absorbance measurements and time-resolved X-ray crystallographic measurements. Trp insertions have been used to block, one at a time, the three major cavities occupied by photodissociated ligands. In this work, we review the effects of the L29(B10)W mutation, which places a large indole ring in the initial 'docking site' for photodissociated ligands. Then, the effects of blocking the Xe4 site with I28W, V68W, and I107W mutations and the Xe1 cavity with L89W, L104W, and F138W mutations are described. The structures of four of these mutants are shown for the first time (Trp28, Trp68, Trp107, and Trp 138 sperm whale metMb). All available results support a 'side path' mechanism in which ligands move into and out of myoglobin by outward rotation of the HisE7 side chain, but after entry can migrate into internal cavities, including the distal Xe4 and proximal Xe1 binding sites. The distal cavities act like the pocket of a baseball glove, catching the ligand and holding it long enough for the histidine gate to close and facilitate internal coordination with the heme iron atom. The physiological role of the proximal Xe1 site is less clear because changes in the size of this cavity have minimal effects on overall O(2) binding parameters. 相似文献
11.
Ligand migration in human myoglobin: steric effects of isoleucine 107(G8) on O(2) and CO binding
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To investigate the ligand pathway in myoglobin, some mutant myoglobins, in which one of the amino acid residues constituting a putative ligand-docking site, Ile107, is replaced by Ala, Val, Leu, or Phe, were prepared and their structural and ligand binding properties were characterized. The kinetic barrier for the ligand entry to protein inside was lowered by decreasing the side-chain volume at position 107, indicating that the bulky side chain interferes with the formation of the activation state for the ligand migration and the free space near position 107 would be filled with the ligand in the activation state. Another prominent effect of the reduced side-chain volume at position 107 is to stabilize the ligand-binding intermediate state. Because the stabilization can be ascribed to decrease of the positive enthalpy, the enlarged free space near position 107 would relieve unfavorable steric interactions between the ligand and nearby amino acid residues. The side-chain volume at position 107, therefore, is crucial for the kinetic barrier for the ligand migration and free energy of the ligand-binding intermediate state, which allows us to propose that some photodissociated O(2) moves toward position 107 to be trapped and then expelled to the solvent. 相似文献
12.
Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-tryptophan (L-Trp) and other indoleamine derivatives. Its activity follows typical Michaelis-Menten behavior only for L-Trp concentrations up to 50 μM; a further increase in the concentration of L-Trp causes a decrease in the activity. This substrate inhibition of hIDO is a result of the binding of a second L-Trp molecule in an inhibitory substrate binding site of the enzyme. The molecular details of the reaction and the inhibition are not yet known. In the following, we summarize the present knowledge about this heme enzyme. 相似文献
13.
Zeqi Li Yuqian Wang Xiaoyan Li Zhenping Lin Yuexu Lin Ramanathan Srinivasan Xiangmin Lin 《Environmental microbiology》2019,21(12):4614-4628
Although many typical outer-membrane proteins (OMPs) have been well characterized, the biological functions of many OMPs remain largely elusive. In this study, we successfully constructed 29 OMP knockout strains in the pathogen Aeromonas hydrophila, which account for about 50% of all predicted OMPs in this bacterial species. We then further validated the antibiotics' susceptibility characteristics against 20 antimicrobial reagents in these mutants considering several phenotypes. Our results showed that a total of 22 OMP mutants affected the susceptibility to at least one antibiotic. The deletion of some OMPs, such as ΔlamB and ΔbamA, revealed very important roles in the resistance to certain antibiotics. However, not a single OMP mutant presented a constant behaviour to all of the tested antibiotics, suggesting the existence of a complex intercellular regulation mechanism and a protein–protein interaction network underlying the OMP homeostasis in the presence of antibiotics. Meanwhile, some OMP mutants also affected biofilm formation, ECPase and haemolytic activity, and carbon resources utilization. This report demonstrates the biological functions of OMPs on a large scale and most of results have not been reported in A. hydrophila. 相似文献
14.
Liver fatty acid-binding protein (FABP) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, and bile acids. Previously we prepared charge reversal mutants and demonstrated the importance of lysine residues within the portal region in ligand and membrane binding. We have now prepared several tryptophan-containing mutants within the portal region, and one tryptophan at position 28 (L28W) has proved remarkably effective as an intrinsic probe to further study ligand binding. The fluorescence of the L28W mutant was very sensitive to fatty acid and bile acid binding where a large (up to 4-fold) fluorescence enhancement was obtained. In contrast, the binding of oleoyl CoA reduced tryptophan fluorescence. Positive cooperativity for fatty acid binding was observed while detailed information on the orientation of binding of bile acid derivatives was obtained. The ability of bound oleoyl CoA to reduce the fluorescence of L28W provided an opportunity to demonstrate that fatty acyl CoAs can compete with fatty acids for binding to liver FABP under physiological conditions, further highlighting the role of fatty acyl CoAs in modulating FABP function in the cell. 相似文献
15.
Bisht NK Abbruzzetti S Uppal S Bruno S Spyrakis F Mozzarelli A Viappiani C Kundu S 《Biochimica et biophysica acta》2011,1814(8):1042-1053
Type 1 non-symbiotic rice hemoglobin (rHb1) shows bis-histidyl heme hexacoordination and is capable of binding diatomic ligands reversibly. The biological function is as yet unclear, but the high oxygen affinity makes it unlikely to be involved in oxygen transport. In order to gain insight into possible physiological roles, we have studied CO rebinding kinetics after laser flash photolysis of rHb1 in solution and encapsulated in silica gel. CO rebinding to wt rHb1 in solution occurs through a fast geminate phase with no sign of rebinding from internal docking sites. Encapsulation in silica gel enhances migration to internal cavities. Site-directed mutagenesis of FB10, a residue known to have a key role in the regulation of hexacoordination and ligand affinity, resulted in substantial effects on the rebinding kinetics, partly inhibiting ligand exit to the solvent, enhancing geminate rebinding and enabling ligand migration within the internal cavities. The mutation of HE7, one of the histidyl residues involved in the hexacoordination, prevents hexacoordination, as expected, but also exposes ligand migration through a complex system of cavities. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. 相似文献
16.
Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization. 相似文献
17.
BA Haines DA Davis A Zykovich B Peng R Rao SD Mooney K Jin DA Greenberg 《Journal of neurochemistry》2012,123(1):192-198
Neuroglobin is a hypoxia-inducible O(2) -binding protein with neuroprotective effects in cell and animal models of stroke and Alzheimer's disease. The mechanism underlying neuroglobin's cytoprotective action is unknown, although several possibilities have been proposed, including anti-oxidative and anti-apoptotic effects. We used affinity purification-mass spectrometry methods to identify neuroglobin-interacting proteins in normoxic and hypoxic murine neuronal (HN33) cell lysates, and to compare these interactions with those of a structurally and functionally related protein, myoglobin. We report that the protein interactomes of neuroglobin and myoglobin overlap substantially and are modified by hypoxia. In addition, neuroglobin-interacting proteins include partners consistent with both anti-oxidative and anti-apoptotic functions, as well as with a relationship to several neurodegenerative diseases. 相似文献
18.
19.
Ligand orientation of oxyproto- and oxymesocobalt porphyrin-substituted myoglobin by single crystal EPR spectroscopy 总被引:1,自引:0,他引:1
Single crystals of oxyproto- and oxymesocobalt myoglobin have been examined by electron paramagnetic resonance spectroscopy at ambient and cryogenic temperatures in order to determine the principal values and eigenvectors of g tensors and the hyperfine coupling tensors. The Co--O--O bond angle was determined to be 125 degrees +/- 5 degrees for oxyprotocobalt myoglobin, and 153 degrees +/- 5 degrees for oxymesocobalt myoglobin at ambient temperature. This result suggests that differences in stereochemical interactions of the modified 2,4-side chains of porphyrin with protein contribute to the ligand orientations as well as the altered ligand-binding behavior in these hemoproteins. Upon freezing, two unequivalent orientations of the O--O axis (species I and II) were found in both oxycobalt myoglobin single crystals. Shifts of the resonance spectra of these species were observed below the freezing point of the crystals. The signal intensities of two paramagnetic species in oxyprotocobalt myoglobin were approximately equivalent (I congruent to II), whereas those in oxymesocobalt myoglobin were quite different (I greater than II) at 77 K. The present electron paramagnetic resonance studies demonstrate that changes in the bonding structure of Co--O2 are induced upon freezing the biological macromolecule, including the movement of the residues of the heme environment. 相似文献
20.
Alan E. Wheals William D. Grant Brigitte M. Jockusch 《Molecular & general genetics : MGG》1976,149(1):111-114
Summary A replica plating method for isolating ts amoebal mutants of Physarum polycephalum has been devised. Temperature-sensitive mutations occur at a frequency after nitrosoguanidine mutagenesis of 10-3 per survivor, are stable but are not usually expressed in the plasmodia formed from these amoebae in clones. Some of these mutants appear to be cell-cycle stage specific. 相似文献