Protein dynamics: conformational disorder,vibrational coupling and anharmonicity in deoxy-hemoglobin and myoglobin

In this work we study the temperature dependence of the Soret band lineshape of deoxymyoglobin and deoxyhemoglobin, in the range 300–20 K. To fit the measured spectra we use an approach originally proposed by Champion and coworkers (Srajer et al. 1986; Srajer and Champion 1991). The band profile is modelled as a Voigt function that accounts for the coupling with low frequency vibrational modes, whereas the coupling with high frequency modes is responsible for the vibronic structure of the spectra. Moreover, owing to the position of the iron atom out of the mean heme plane, inhomogeneous broadening brings about a non-Gaussian distribution of 0–0 electronic transition frequencies. The reported analysis enables us to isolate the various contributions to the overall bandwidth, and their temperature dependence points out the relevance of low frequency vibrations and of large scale anharmonic motions starting at temperatures higher than 170 K. Information on the mean iron-heme plane distance and on its temperature dependence, as well as on the heme pocket conformational disorder, is also obtained.Abbreviations Cc Carbon monoxide - Hb Human deoxyhemoglobin A - HbCO human carbonmonoxyhemoglobin A - SWMb spermwhale deoxymyoglobin - SWMbCO spermwhale carbonmonoxymyoglobin - HbO₂ human oxyhemoglobin A - SWMb³⁺-H₂O spermwhale aquometmyoglobin     

Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed. Correspondence to: R. Rigler     

Long-distance cofactor interactions in terminal oxidases studied by second-derivative absorption spectroscopy

The electronic transitions of the two heme groups of cytochromec oxidase have been resolved by application of second-derivative and cryogenic absorption spectroscopy. Both methods reveal a splitting of the ferrocytochromea Soret transition into two features at 443 and 450 nm. The relative intensity of the 450 nm feature appears to depend on the ligation state of cytochromea ₃, the solution pH, and complex formation with cytochromec. The structural origin and mechanistic significance of this second Soret transition of cytochromea are discussed in terms of the electron transfer and proton translocation activities of the enzyme.Dedicated to the memory of James Carl Copeland.     

Mouse polyomavirus mediated effects on the infected cell membrane studied by dielectric spectroscopy

The effect of polyomavirus on the infected cell has been investigated by dieclectric spectroscopy. This technique has a great potential in the study of the ion transport properties of the cell membrane. The results presented in this communication suggest a correlation between progression of the viral infection and dielectric features of the infected, cell plasma membrane.     

Discrete steps in dioxygen activation—The cytochrome oxidase/O2 reaction

The kinetic constraints that are imposed on cytochrome oxidase in its dual function as the terminal oxidant in the respiratory process and as a redox-linked proton pump provide a unique opportunity to investigate the molecular details of biological O₂ activation. By using flow/flash techniques, it is possible to visualize individual steps in the O₂-binding and reduction process, and results from a number of spectroscopic investigations on the oxidation of reduced cytochrome oxidase by O₂ are now available. In this article, we use these results to synthesize a reaction mechanism for O₂ activation in the enzyme and to simulate time-concentration profiles for a number of intemediates that have been observed experimentally. Kinetic manifestation of the consequences of coupling exergonic electron transfer to endergonic proton translocation emerge from this analysis. Energetic efficiency in this process apparently requires that potentially toxic intermediate oxidation states of dioxygen accumulate to substantial concentration during the reduction reaction.     

Neither Moderate Hypoxia nor Mild Hypoglycaemia Alone Causes Any Significant Increase in Cerebral [Ca2±i: Only a Combination of the Two Insults Has This Effect.: A 31P and 19F NMR Study

(1) The energy state and free intracellular calcium concentration ([Ca^2±_i) of super-fused cortical slices were measured in moderate hypoxia (～65 μM O₂), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using ¹⁹F and ³¹P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca^2±_i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca^2±, and a decrease in PCr level to ～25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca^2±_i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca^2±_i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca^2±_i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult.     

乌骨鸡黑素的一些基本结构特征的初步研究

应用元素分析、电子能谱(XPS)和红外光谱(IR)分析技术对乌骨鸡体内黑素结构特征进行了研究。元素分析结果得到黑素分子中C、H、N、O克原子比率的均值为10:12:1:3.该比值与动物性吲哚黑素的相应元素比例接近。XPS波谱显示出黑素中含有两种价态的硫(S~Ⅵ、S~Ⅰ),其中以低价态占优势,S2p电子结合能比人发黑素要大些。氮原子只有一种价态。IR光谱中有五组明显吸收峰,其中以主要表征吲哚环的1619—1632CM~(-1)处的吸引峰最强。以上结果初步表明出乌骨鸡黑素是以吲哚环为主体的异聚物。     

Evidence for two different blue-light-receptor systems for the fast responses of stimulation of photosynthetic capacity and acidification of the plant surface in brown algae

Two blue-light responses of Phaeophyta that are expressed within a few seconds of a blue-light stimulus were characterized with respect to their photoreception properties. The first response is the activation of red-light-saturated photosynthesis which can be stimulated to values up to 5 times the rates in red light, depending on the species. The second response is a blue-light-induced acidification measurable at the plant surface. Both responses have similar kinetic characteristics and thus led us initially to hypothesise that they were causally connected in the same transduction mechanism. The two responses have action spectra [measured for Ectocarpus siliculosus (Dillwyn) Lyngb. and Laminaria saccharina (L.) Lamouroux] that are indistinguishable within the relatively large limits of error. However, in all species tested, the threshold sensitivity for blue light of the photosynthetic response is lower than that of the pH-shift by a factor of 2 to 150. Furthermore, stimulation of photosynthesis is sensitive to the flavin inhibitors, KI and phenylacetic acid, but the pH response is not affected by these inhibitors. Thus, the blue-light-induced pH-shift does not cause the stimulation of photosynthesis. In contrast, the different fluence-response relationships of the two responses and particularly the differential effect of the inhibitors are clear evidence for the action of two independent transduction pathways and photoreceptor systems for blue light. At least photoreception for stimulation of photosynthesis involves a flavin-or and a pterin.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PAA phenylacetic acid We thank Dr. C. A. Maggs for collecting P. pavonica. This research was supported by National Environment Research Council grant No. GR3/8102.     

31P-Nuclear magnetic resonance spectroscopy study of the time course of energy metabolism during exercise and recovery

This study evaluated the time courses of intracellular pH and the metabolism of phosphocreatine (PCr) and inorganic phosphate (P) at the onset of four exercise intensities and recoveries. Non-invasive evaluation of continuous changes in phosphorus metabolites has become possible using³¹P-nuclear magnetic resonance spectroscopy (³¹P-MRS). After measurements at rest, six healthy male subjects performed 4 min of femoral flexion exercise at intensities of 0 (loadless), 10, 20 and 30 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore. Measurements were continuously made during 5 min of recovery. During a series of rest-exercise-recovery procedures,³¹P-MRS were accumulated using 32 scans · spectrum^–1 requiring 12.8 s each. At the onset of exercise, PCr decreased exponentially with a time constant of 27–32 s regardless of the exercise intensity. The time constant PCr resynthesis during recovery was about 27–40 s. The PCr kinetics were independent of exercise intensity. There were similar Pi kinetics at the onset of all types of exercise, while those of Pi recovery became significantly longer at the higher exercise intensities (P < 0.05). Furthermore, the intracellular pH indicated temporary alkalosis just at the onset of exercise, probably due to absorption of hydrogen ions by PCr hydrolysis, and then decrease at a point about 40%–50% of the preexercise PCr. The pH recovery time was longer than that for the P_i or PCr kinetics. By using a more efficient resolution system it was possible to obtain the phosphorus kinetics during exercise and to follow PCr resynthesis within the first few minutes of recovery. From our results it was concluded that in general the time course of PCr and Pi metabolism were unaffected by the exercise intensity, both at the onset of exercise and during recovery, with the exception of P_i recovery.     

Changes in intracellular pH during repeated exercise

The rates of change in intracellular pH during repeated exercise sessions with rest periods was determined by 31 phosphorus-nuclear magnetic resonance spectroscopy (³¹P-MRS). Five long-distance runners and six healthy male subjects as controls performed a 2-min femoral flexion at 20 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore and repeated this exercise four times with 2-min rest periods intervening. In all cases during exercise the inorganic phosphate (P_i) peak split into two, the earlier increased rapidly (high-pH P_i) and the later (low-pH P_i) increased more slowly. The P_i peaks were separated by a fitting procedure using the least square mean method. The high-pH P_i area during exercise decreased as the number of repeated exercise periods increased, while the low-pH P_i area gradually increased. Although the total P_i area decreased exponentially during the recovery period, the high-pH P_i area decreased first and then the low-pH P_i area reduced gradually. The pH values were estimated from the chemical shift between the phosphocreatine peak and each split peak in the P_i. The high-pH in pooled data ranged from 6.6 to 7.0 during exercise and recovery, while the low pH decreased to 6.2 during exercise. As the number of exercise periods increased, each pH value gradually became less acidic, although there was a tendency to more acidity in the control subjects than in the long-distance runners. In conclusion, it was possible to obtain by non-invasive, continuous³¹P-MRS, a split pattern of P_i peaks during exercise and there were at least tow different intracellular pH values during exercise, suggesting that each P_i peak might be attributed to the types of muscle fibre recruited.