Temperature dependence of fast carbonyl backbone dynamics in chicken villin headpiece subdomain

Temperature-dependence of protein dynamics can provide information on details of the free energy landscape by probing the characteristics of the potential responsible for the fluctuations. We have investigated the temperature-dependence of picosecond to nanosecond backbone dynamics at carbonyl carbon sites in chicken villin headpiece subdomain protein using a combination of three NMR relaxation rates: ¹³C′ longitudinal rate, and two cross-correlated rates involving dipolar and chemical shift anisotropy (CSA) relaxation mechanisms, ¹³C′/¹³C′-¹³C^α CSA/dipolar and ¹³C′/¹³C′–¹⁵N CSA/dipolar. Order parameters have been extracted using the Lipari-Szabo model-free approach assuming a separation of the time scales of internal and molecular motions in the 2–16°C temperature range. There is a gradual deviation from this assumption from lower to higher temperatures, such that above 16°C the separation of the time scales is inconsistent with the experimental data and, thus, the Lipari-Szabo formalism can not be applied. While there are variations among the residues, on the average the order parameters indicate a markedly steeper temperature dependence at backbone carbonyl carbons compared to that probed at amide nitrogens in an earlier study. This strongly advocates for probing sites other than amide nitrogen for accurate characterization of the potential and other thermodynamics characteristics of protein backbone.     

Temperature dependence of partial volumes of proteins

The change of the apparent partial specific volumes of egg albumin, bovine serum albumin, bovine methemoglobin, β-lactoglobulin, and lysozyme with temperature through the thermal transitions of the proteins have been measured with dilatometers. Four regions in the plot of the apparent partial specific volumes against temperature can be recognized: (1) linear sections extending from 25°C up to 45–50°C: (2) a decrease in slope between 50°C and 60°C; (3) a sharp increase in slope with increasing temperature coinciding with the appearance of heat coagulation of the protein and followed by (4) a decrease in the slope. The return of the protein samples to 25°C yields linear relations between the apparent partial specific volumes of the heat-denatured proteins and the decreasing temperature.     

Temperature dependence of 1H chemical shifts in proteins

Temperature coefficients have been measured by 2D NMR methods forthe amide and CH proton chemical shifts in two globularproteins, bovine pancreatic trypsin inhibitor and hen egg-white lysozyme.The temperature-dependent changes in chemical shift are generally linear upto about 15° below the global denaturation temperature, and the derivedcoefficients span a range of roughly –16 to +2 ppb/K for amide protonsand –4 to +3 ppb/K for CH. The temperaturecoefficients can be rationalized by the assumption that heating causesincreases in thermal motion in the protein. Precise calculations oftemperature coefficients derived from protein coordinates are not possible,since chemical shifts are sensitive to small changes in atomic coordinates.Amide temperature coefficients correlate well with the location of hydrogenbonds as determined by crystallography. It is concluded that a combined useof both temperature coefficients and exchange rates produces a far morereliable indicator of hydrogen bonding than either alone. If an amide protonexchanges slowly and has a temperature coefficient more positive than–4.5 ppb/K, it is hydrogen bonded, while if it exchanges rapidly andhas a temperature coefficient more negative than –4.5 ppb/K, it is nothydrogen bonded. The previously observed unreliability of temperaturecoefficients as measures of hydrogen bonding in peptides may arise fromlosses of peptide secondary structure on heating.     

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

The dynamics of water molecules near the protein surface are different from those of bulk water and influence the structure and dynamics of the protein itself. To elucidate the temperature dependence hydration dynamics of water molecules, we present results from the molecular dynamic simulation of the water molecules surrounding two proteins (Carboxypeptidase inhibitor and Ovomucoid) at seven different temperatures (T=273 to 303 K, in increments of 5 K). Translational diffusion coefficients of the surface water and bulk water molecules were estimated from 2 ns molecular dynamics simulation trajectories. Temperature dependence of the estimated bulk water diffusion closely reflects the experimental values, while hydration water diffusion is retarded significantly due to the protein. Protein surface induced scaling of translational dynamics of the hydration waters is uniform over the temperature range studied, suggesting the importance protein-water interactions.     

Temperature dependence of the internal dynamics of a calmodulin-peptide complex

The temperature dependence of the fast internal dynamics of calcium-saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase is examined using 15N and 2H NMR relaxation methods. NMR relaxation studies of the complex were carried out at 13 temperatures that span 288-346 K. The dynamics of the backbone and over four dozen methyl-bearing side chains, distributed throughout the calmodulin molecule, were probed. The side chains show a much more variable and often considerably larger response to temperature than the backbone. A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. The amplitude of motion of some side chains is essentially temperature-independent while many show a simple roughly linear temperature dependence. In a few cases, angular order increases with temperature, which is interpreted as arising from interactions with neighboring residues. In addition, a number of side chains display a nonlinear temperature dependence. The significance of these and other results is illuminated by several simple interpretative models. Importantly, analysis of these models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions largely arises from local bond vibrations and solvent interactions and not from torsional oscillations of side chains.     

Temperature dependence of MinD oscillation in Escherichia coli: running hot and fast

We observed that the oscillation period of MinD within rod-like and filamentous cells of Escherichia coli varied by a factor of 4 in the temperature range from 20 degrees C to 40 degrees C. The detailed dependence was Arrhenius, with a slope similar to the overall temperature-dependent growth curve of E. coli. The detailed pattern of oscillation, including the characteristic wavelength in filamentous cells, remained independent of temperature. A quantitative model of MinDE oscillation exhibited similar behavior, with an activated temperature dependence of the MinE-stimulated MinD-ATPase rate.     

Temperature dependence of vesicular dynamics at excitatory synapses of rat hippocampus

How vesicular dynamics parameters depend on temperature and how temperature affects the parameter change during prolonged high frequency stimulation was determined by fitting a model of vesicular storage and release to the amplitudes of the excitatory post-synaptic currents (EPSC) recorded from CA1 neurons in rat hippocampal slices. The temperature ranged from low (13 °C) to higher and more physiological temperature (34 °C). Fitting the model of vesicular storage and release to the EPSC amplitudes during a single pair of brief high–low frequency stimulation trains yields the estimates of all parameters of the vesicular dynamics, and with good precision. Both fractional release and replenishment rate decrease as the temperature rises. Change of the underlying ‘basic’ parameters (release coupling, replenishment coupling and readily releasable pool size), which the model-fitting also yields is complex. The replenishment coupling between the readily releasable pool (RRP) and resting pool increases with temperature (which renders the replenishment rate higher), but this is more than counterbalanced by greater RRP size (which renders the replenishment rate lower). Finally, during long, high frequency patterned stimulation that leads to significant synaptic depression, the replenishment rate decreases markedly and rapidly at low temperatures (<22 °C), but at high temperatures (>28 °C) the replenishment rate rises with stimulation, making synapses better able to maintain synaptic efficacy.     

Temperature dependence of rotational dynamics of protein and lipid in sarcoplasmic reticulum membranes

We have investigated the relationship between function and molecular dynamics of both the lipid and the Ca-ATPase protein in sarcoplasmic reticulum (SR), using temperature as a means of altering both activity and rotational dynamics. Conventional and saturation-transfer electron paramagnetic resonance (EPR) was used to probe rotational motions of spin-labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase sulfhydryl groups in SR. EPR studies were also performed on aqueous dispersions of extracted SR lipids, in order to study intrinsic lipid properties independent of the protein. While an Arrhenius plot of the Ca-ATPase activity exhibits a clear change in slope at 20 degrees C, Arrhenius plots of lipid hydrocarbon chain mobility are linear, indicating that an abrupt thermotropic change in the lipid hydrocarbon phase is not responsible for the Arrhenius break in enzymatic activity. The presence of protein was found to decrease the average hydrocarbon chain mobility, but linear Arrhenius plots were observed both in the intact SR and in extracted lipids. Lipid EPR spectra were analyzed by procedures that prevent the production of artifactual breaks in the Arrhenius plots. Similarly, using sample preparations and spectral analysis methods that minimize the temperature-dependent contribution of local probe mobility to the spectra of spin-labeled Ca-ATPase, we find that Arrhenius plots of overall protein rotational mobility also exhibit no change in slope. The activation energy for protein mobility is the same as that of ATPase activity above 20 degrees C; we discuss the possibility that overall protein mobility may be essential to the rate-limiting step above 20 degrees C.     

Temperature dependence of the partition coefficient of proteins in aqueous two-phase systems.

We report the partition coefficients of lysozyme, chymotrypsinogen-A, albumin and catalase in sixty four Polyethyleneglycol/Dextran/Water systems at 4, 25 and 40 degrees C. We found that the partition coefficients of the four proteins generally increase with increasing temperature. The influence of temperature on the partition coefficient seems to be highly dependent on the kind of protein which is partitioned and on the total polymer concentration, but does not, in general, depend on the molecular weight of the polymers. The partition coefficients of small and hydrophilic proteins like lysozyme and chymotrypsinogen-A are only slightly affected by changes in temperature, while the partition coefficients of bigger and more hydrophobic proteins like albumin and catalase are strongly affected by changes in temperature. The results suggest the incorporation of attractive forces (possible electrostatic) into a model previously reported by us.     

Temperature dependence of the inhibitory effects of orthovanadate on shortening velocity in fast skeletal muscle.

We have investigated the effects of the orthophosphate (P(i)) analog orthovanadate (Vi) on maximum shortening velocity (Vmax) in activated, chemically skinned, vertebrate skeletal muscle fibers. Using new "temperature-jump" protocols, reproducible data can be obtained from activated fibers at high temperatures, and we have examined the effect of increased [Vi] on Vmax for temperatures in the range 5-30 degrees C. We find that for temperatures < or = 20 degrees C, increasing [Vi] inhibits Vmax; for temperatures > or = 25 degrees C, increasing [Vi] does not inhibit Vmax. Attached cross-bridges bound to Vi are thought to be an analog of the weakly bound actin-myosin.ADP-P(i) state. The data suggest that the weakly bound Vi state can inhibit velocity at low temperature, but not at high temperature, with the transition occurring over a narrow temperature range of < 5 degrees C. This suggests a highly cooperative interaction. The data also define a Q10 for Vmax of 2.1 for chemically skinned rabbit psoas fibers over the temperature range of 5-30 degrees C.     

Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins

The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.     

Temperature dependence of cytochrome photooxidation and conformational dynamics of Chromatium reaction center complexes

A temperature dependence of multiheme cytochrome c oxidation induced by a laser pulse was studied in photosynthetic reaction center preparations from Chromatium minutissimum. Absorbance changes and kinetic characteristics of the reaction were measured under redox conditions where one or all of the hemes of the cytochrome subunit are chemically reduced (E _h =+300 mV or E _h =–20 to -60 mV respectively). In the first case photooxidation is inhibited at temperatures lower than 190–200 K with the rate constant of the photooxidation reaction being practically independent on temperature over the range of 300 to 190 K (k=2.2×10⁵ s^-1). Under reductive conditions (E _h =–20 to -60 mV) lowering the temperature to 190–200 K causes the reaction to slow from k=8.3×10⁵ s^-1 to 2.1×10⁴ s^-1. Under further cooling down to the liquid nitrogen temperature, the reaction rate changes negligibly. The absorption amplitude decreases by 30–40% on lowering the temperature. A new physical mechanism of the observed critical effects of temperature on the rate and absorption amplitude of the multiheme cytochrome c oxidation reaction is proposed. The mechanism suggests a close interrelation between conformational mobility of the protein and elementary electron tunneling act. The effect of freezing conformational motion is described in terms of a local diffusion along a random rough potential.     

Temperature dependence of the structure and dynamics of myoglobin. A simulation approach

The results of simulations of the structure and internal motions of carbonomonoxymyoglobin (MbCO) at two different temperatures (325 and 80 K) are presented and compared with experimental data. Properties calculated from the 120 ps trajectory at 325 K are used as a reference in the analysis of the motion of the protein at 80 K. Three separate 80 K molecular dynamics trajectories were calculated; they were started with different coordinate sets from the 325 K simulation and the lower temperature was achieved by scaling the velocities. The simulations yield results for the structural changes between 325 and 80 K that are in general accord with those from X-ray data. Both the experimental and calculated radii of gyration, distances from the center of mass and main-chain difference distance matrices show that there is a significant but inhomogeneous shrinkage with decreasing temperature. For the atomic fluctuations, by contrast, the calculated temperature dependence is very different from the X-ray results; i.e. the calculated root-mean-square backbone fluctuations decrease to 0.11 A at 80 K from 0.51 A at 325 K, while the fluctuations obtained from the X-ray B factors go from 0.56 A at 260 K to 0.47 A at 80 K. The smaller temperature dependence of the B factors suggests that there is significant conformational disorder in MbCO crystals at lower temperatures. This is in accord with the simulation results, which show that the protein is trapped in restricted regions of conformational space at 80 K, while at 325 K a much larger region is accessible to the protein. Analysis of the fluctuations at 325 K and 80 K shows that the room temperature flexibility of the protein is determined by the mobility of the loop regions and by side-chain torsional motions (in accord with earlier simulation results), while the low temperature fluctuations involve motion within a single well. Examination of the calculated iron atom fluctuations and comparison with Mossbauer data show good agreement. It is found that the dominant contribution to the iron motion arises from heme sliding; motion of the iron relative to the heme are much smaller.     

Temperature dependence of protein dynamics: computer simulation analysis of neutron scattering properties

The temperature dependence of the internal dynamics of an isolated protein, bovine pancreatic trypsin inhibitor, is examined using normal mode analysis and molecular dynamics (MD) simulation. It is found that the protein exhibits marked anharmonic dynamics at temperatures of approximately 100-120 K, as evidenced by departure of the MD-derived average mean square displacement from that of the harmonic model. This activation of anharmonic dynamics is at lower temperatures than previously detected in proteins and is found in the absence of solvent molecules. The simulation data are also used to investigate neutron scattering properties. The effects are determined of instrumental energy resolution and of approximations commonly used to extract mean square displacement data from elastic scattering experiments. Both the presence of a distribution of mean square displacements in the protein and the use of the Gaussian approximation to the dynamic structure factor lead to quantified underestimation of the mean square displacement obtained.     

Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins.

A hallmark of alpha-crystallin-type small heat shock proteins (sHsps) is their highly dynamic oligomeric structure which promotes intermolecular interactions involved in subunit exchange and substrate binding (chaperone-like activity). We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectrospray mass spectrometry. Proteins of both classes formed large complexes that rapidly dissociated upon dilution and at physiologically relevant heat shock temperatures. As the secondary structure was not perturbed, temperature- and concentration-dependent dissociations were fully reversible. Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers. Small Hsps, mutated in a highly conserved glycine residue at the C-terminal end of the alpha-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins. Reduced complex stability coincided with reduced chaperone activity.     

Temperature dependence of speed of actin filaments propelled by slow and fast skeletal myosin isoforms.

It was shown that the temperature sensitivity of shortening velocity of skeletal muscles is higher at temperatures below physiological (10-25 degrees C) than at temperatures closer to physiological (25-35 degrees C) and is higher in slow than fast muscles. However, because intact muscles invariably express several myosin isoforms, they are not the ideal model to compare the temperature sensitivity of slow and fast myosin isoforms. Moreover, temperature sensitivity of intact muscles and single muscle fibers cannot be unequivocally attributed to a modulation of myosin function itself, as in such specimen myosin works in the structure of the sarcomere together with other myofibrillar proteins. We have used an in vitro motility assay approach in which the impact of temperature on velocity can be studied at a molecular level, as in such assays acto-myosin interaction occurs in the absence of sarcomere structure and of the other myofibrillar proteins. Moreover, the temperature modulation of velocity could be studied in pure myosin isoforms (rat type 1, 2A, and 2B and rabbit type 1 and 2X) that could be extracted from single fibers and in a wide range of temperatures (10-35 degrees C) because isolated myosin is stable up to physiological temperature. The data show that, at the molecular level, the temperature sensitivity is higher at lower (10-25 degrees C) than at higher (25-35 degrees C) temperatures, consistent with experiments on isolated muscles. However, slow myosin isoforms did not show a higher temperature sensitivity than fast isoforms, contrary to what was observed in intact slow and fast muscles.     

Backbone dynamics, fast folding, and secondary structure formation in helical proteins and peptides

We discuss the construction of a simple, off-lattice model protein with a comparatively detailed representation of the protein backbone, and use it to address some general aspects of the folding kinetics of a small helical protein and two peptide fragments. The model makes use of an associative memory hamiltonian to smoothly interpolate between the limits of a native contact only, or Go, potential and a statistical pair potential derived from a database of known structures. We have observed qualitatively different behavior in these two limits. In the Go limit, we see apparently barrier-less folding. As we increase the roughness of the model energy landscape, we can observe the emergence of the characteristic activated temperature dependence previously seen in lattice studies and analytical theories. We are also able to study the dependence of the folding kinetics on local interactions such as hydrogen bonds, and we discuss the implications of these results for the formation of secondary structure at intermediate stages of the folding reaction.     

Temperature dependence of photosynthesis in cotton

Cotton plants (Gossypium hirsutum L., var. Deltapine Smooth Leaf) were grown under controlled environmental conditions over a range of day/night temperatures from 20/15 to 40/35 C. Their photosynthetic characteristics were then measured over a comparable temperature range. Net photosynthesis tended stongly to be greatest, and intracellular resistance to CO₂ transport to be lowest, when the measurement temperature corresponded to the daytime growth temperature, suggesting pronounced acclimation of the plants to the growth temperature. The preferred growth temperature was close to the 25/20 C regime, since net photosynthesis of these plants, regardless of measurement temperature, was higher and intracellular resistance lower, than in plants from any other regime.