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Instrumentation
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
The temperature-jump reaction cell itself is thermally insulated so that, following the rapid increase in temperature, the reactants remain at the elevated temperature for a short period, milliseconds to seconds depending on the volume of the cell. Here, the design of the apparatus is a compromise between holding the conditions stable, while the transient response is monitored, and allowing the solutions to cool back to their original temperature so that the experiment may be repeated to achieve the benefit of signal averaging. It is possible to incorporate supplementary switchable heating and cooling circuit using water flow or Peltier elements to hold the cell at the constant elevated temperature until the reaction is complete and then rapidly cool it. However, temperature jump is usually employed to study reactions on the millisecond time scale or less so that this sophistication is not generally necessary.
Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
The time course of the heat-induced protein unfolding or misfolding was monitored in kinetic temperature-jump (T-jump) experiments. The unfolding was triggered at time t=0 by a rapid temperature increase from 25oC to a new constant value (37-70oC), and was monitored by CD at 202 nm, 6202(t), to optimize the signal-to-noise ratio.
The Reactivity Of Copper Sites In The “Blue” Copper Proteins
Published in René Lontie, Copper Proteins and Copper Enzymes, 1984
The electron-transfer reaction between Pseudomonas Αz and Cyt c55] has been intensively studied.53-59 In the first investigation carried out by stopped-flow, it was shown that the second-order rate constant reached a limiting value at high Αz concentration.53 This finding indicated that the reaction is not a simple reversible electron-transfer process between the two partners, but rather suggested a possible complex formation. In the later temperature-jump kinetic studies, two distinct relaxation times were reported, and careful analysis of both kinetic and thermodynamic parameters led to the conclusion that complex formation was insignificant.54,55 The reciprocal value of the faster relaxation time showed a linear increase up to high Αz concentration with no tendency to level off, whereas the slower one displayed a limiting dependence on Αz concentration. Based on these observations it was suggested that Αz(1) exists in two different conformers, one of which is inactive and does not participate in electron transfer.54,55 In addition, because of an apparent discrepancy between the enthalpy of the electron-transfer equilibrium, and the observed dependence of this reaction on temperature, a further endothermic conformational equilibrium of the oxidized Cyt ί·55| was postulated. This yielded the following scheme:55
An additive destabilising effect of compound T60I and V122I substitutions in ATTRv amyloidosis
Published in Amyloid, 2023
Tatiana Prokaeva, Elena S. Klimtchuk, Polina Feschenko, Brian Spencer, Haili Cui, Eric J. Burks, Roshanak Aslebagh, Khaja Muneeruddin, Scott A. Shaffer, Elizabeth Varghese, John L. Berk, Lawreen H. Connors
The recombinant T60I and V122I proteins were mixed in varying amounts (3:1, 1:1, 1:3 ratios) at a total TTR concentration of 0.2 mg/mL. Mixtures were incubated at 4 °C for 48 h to allow for subunit exchange [29]. CD spectra, melting and kinetic temperature-jump data were recorded by using Jasco J-815 (Jasco Inc., Japan) spectropolarimeter equipped with a thermoelectric temperature controller. Secondary structure was assessed by far-UV CD spectra recorded at 190–250 nm on samples containing 0.2 mg/mL TTR placed in 1 mm quartz cells (Figure 5(A)). Tertiary structure was assessed by near-UV CD spectra recorded at 250–300 nm on samples containing 0.6 mg/mL protein placed in 10 mm quartz cells (Figure 5(B)). In temperature-jump experiments, the sample was rapidly heated at time t = 0 from 25 °C to 85 °C to trigger protein unfolding; the time course of Trp exposure upon unfolding at 85 °C was monitored by CD at 291 nm for over 20 h (Figure 5(C)). Melting data were recorded at 215 nm to monitor β-sheet unfolding during sample heating from 25 °C to 98 °C at a constant rate of 6 °C/h (Figure 5(D)). The data were normalised to protein concentration and expressed in units of molar residue ellipticity (MRE) for far-UV CD or molar ellipticity (ME) for near-UV CD.
Modeling thermal contact resistance at the finger-object interface
Published in Temperature, 2019
“Don’t touch that, it’s hot!” is a phrase everyone has exclaimed at some point, often to a child who neglects these wise words and proceeds to test the said statement to a painful conclusion. Interestingly, how “hot” or “cold” an object feels not only depends on its temperature in respect to that of the skin, but as recently reviewed by Hsin-Ni Ho [1] in this journal, also on a surprising number of parameters involved in physical, perceptual, and cognitive responses. One common observation that highlights the role of thermophysical properties is that metal objects feel colder than glass or plastics at the same temperature [1,2]. Additionally, we know that, to a certain degree, an object will feel hotter or colder when we press on it harder, which highlights the dependence of skin temperature on imposed contact force, Figure 1(a) illustrates that this temperature jump, 2–6], thermal displays [7], artificial hands [8], haptic devices [9], and wearable thermoelectric devices [10,11], it is important to know how to combine these physical parameters to model the value of
A novel anti-platelet aggregation target of chinensinaphthol methyl ether and neojusticin B obtained from Rostellularia procumbens (L.) Nees
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Songtao Wu, Yanfang Yang, Bo Liu, Zhoutao Xie, Weichen Xiong, Pengfei Hao, Wenping Xiao, Yuan Sun, Zhongzhu Ai, Hezhen Wu
Approximately, 3 µl was loaded into NT.LabelFree standard-treated capillaries (Nanotemper). MST experiments were performed at 40% MST (infra-red laser) power and 60% LED power at 25 °C using the Monolith NT.LabelFree Instrument (Nanotemper, Munich, Germany). Ratios between normalized initial fluorescence and after temperature-jump and thermophoresis were calculated and averaged from 5 to 9 independent runs. Means of fluorescence intensity obtained by the MST measurements were fitted, and the resultant Kd values were given together with an error estimation from the fit by the built-in formula of the analysis software.