scholarly journals Calorimetric studies of the N-terminal half-molecule of transferrin and mutant forms modified near the Fe3+-binding site

1993 ◽  
Vol 293 (2) ◽  
pp. 517-522 ◽  
Author(s):  
L N Lin ◽  
A B Mason ◽  
R C Woodworth ◽  
J F Brandts
Keyword(s):  
Binding Affinity ◽  
Binding Constant ◽  
Conformational Stability ◽  
High Sensitivity ◽  
Single Amino Acid ◽  
Iron Binding ◽  
Scanning Calorimetry ◽  
Apparent Binding Constant ◽  
Binding Cleft ◽  
Calorimetric Studies

The effects of single amino acid substitution on the thermal stability of the N-terminal half-molecule of human transferrin and its iron-binding affinity have been studied by high-sensitivity scanning calorimetry. All site-directed mutations are located on the surface of the binding cleft, and they are D63-->S, D63-->C, G65-->R, H207-->E and K206-->Q. Differential scanning calorimetry results show that the mutations do not significantly alter the conformational stability of the apo-forms of the proteins. The changes in free energy of unfolding relative to the wild-type protein range from 0.83 to -2.4 kJ/mol. The D63-->S, G54-->R and H207-->E mutations slightly destabilize the apo-protein, while the D63-->C and K206-->Q mutations increase its stability by a small amount. However, there are large compensating enthalpy-entropy changes caused by all mutations. All mutants bind ferric ion, but with different affinities. Replacement of Asp-63 by either Ser or Cys decreases the apparent binding constant by 5-6 orders of magnitude. The G65-->R mutation also decreases the apparent binding constant by 5 orders of magnitude. The K206-->Q mutation increases the apparent binding constant by 20-fold, while the H207-->E mutation does not significantly change the apparent iron-binding affinity of the half-molecule.

scholarly journals Calorimetric Studies of Melanotransferrin (p97) and Its Interaction with Iron

2005 ◽  
Vol 280 (16) ◽  
pp. 15735-15741 ◽  
Author(s):  
A. Louise Creagh ◽  
Jacqueline W. C. Tiong ◽  
Mei Mei Tian ◽  
Charles A. Haynes ◽  
Wilfred A. Jefferies
Keyword(s):  
Differential Scanning Calorimetry ◽  
Isothermal Titration Calorimetry ◽  
Binding Constant ◽  
Direct Determination ◽  
Molar Ratio ◽  
Titration Calorimetry ◽  
Iron Binding ◽  
Scanning Calorimetry ◽  
Binding Characteristics ◽  
Apparent Binding Constant

The mammalian molecule melanotransferrin (mTf), also called p97, is a member of the transferrin family of molecules. It exists in both secreted and glycosylphosphatidylinositol-anchored forms and is thought to play a role in angiogenesis and in transporting iron across the blood brain barrier. The binding affinity of iron to this molecule has not been formally established. Here, the binding of ferric ion (chelated with a 2-fold molar ratio of nitrilotriacetate) to mTf has been studied using isothermal titration calorimetry and differential scanning calorimetry. One iron-binding site was determined for mTf with similar binding characteristics to other transferrins. In the absence of bicarbonate, binding occurs quickly with an apparent association constant of 2.6 × 107m–1at 25 °C. The presence of bicarbonate introduces kinetic effects that prevent direct determination of the apparent binding constant by isothermal titration calorimetry. Differential scanning calorimetry thermograms of mTf unfolding in the presence and absence of iron were therefore used to determine the apparent binding constant in the bicarbonate-containing system; at pH 7.5 and 25 °C, iron binding occurs in a 1:1 ratio with aKappof 4.4 × 1017m–1. This affinity is intermediate between the high and low affinity lobes of transferrin and suggests that mTf is likely to play a significant role in iron transport where the high affinity lobe of transferrin is occupied or where transferrin is in proportionally low concentrations.

scholarly journals Competition between Structural Relaxation and Crystallization in the Glass Transition Range of Random Copolymers

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1778
Author(s):  
Jürgen E. K. Schawe ◽  
Claus Wrana
Keyword(s):  
Glass Transition ◽  
Structural Relaxation ◽  
Random Sequence ◽  
High Sensitivity ◽  
Random Copolymers ◽  
High Time Resolution ◽  
Scanning Calorimetry ◽  
Transition Range ◽  
Glass Transition Range ◽  
And Control

Structural relaxation in polymers occurs at temperatures in the glass transition range and below. At these temperatures, crystallization is controlled by diffusion and nucleation. A sequential occurrence of structural relaxation, nucleation, and crystallization was observed for several homopolymers during annealing in the range of the glass transition. It is known from the literature that all of these processes are strongly influenced by geometrical confinements. The focus of our work is copolymers, in which the confinements are caused by the random sequence of monomer units in the polymer chain. We characterize the influence of these confinements on structure formation and relaxation in the vicinity of the glass transition. The measurements were performed with a hydrogenated nitrile-butadiene copolymer (HNBR). The kinetics of the structural relaxation and the crystallization was measured using fast differential scanning calorimetry (FDSC). This technique was selected because of the high sensitivity, the fast cooling rates, and the high time resolution. Crystallization in HNBR causes a segregation of non-crystallizable segments in the macromolecule. This yields a reduction in mobility in the vicinity of the formed crystals and as a consequence an increased amount of so-called “rigid amorphous fraction” (RAF). The RAF can be interpreted as self-assembled confinements, which limit and control the crystallization. An analysis of the crystallization and the relaxation shows that the kinetic of both is identical. This means that the Kohlrausch exponent of relaxation and the Avrami exponent of crystallization are identical. Therefore, the crystallization is not controlled by nucleation but by diffusion and is terminated by the formation of RAF.

scholarly journals Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

2016 ◽  
Vol 90 (19) ◽  
pp. 8542-8551 ◽  
Author(s):  
Lauren M. Drouin ◽  
Bridget Lins ◽  
Maria Janssen ◽  
Antonette Bennett ◽  
Paul Chipman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Intramolecular Interactions ◽  
Single Amino Acid ◽  
Fold Axis ◽  
Wild Type ◽  
Scanning Calorimetry ◽  
Genome Packaging ◽  
Cryo Electron Microscopy ◽  
Empty Capsid ◽  
Packaging Efficiency

ABSTRACTThe adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.IMPORTANCEThe mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

Effect of Concentration of KI and of Temperature on Shortening of Glycerol-Treated Muscle Fibers in KI Solutions

1956 ◽  
Vol 185 (1) ◽  
pp. 92-94 ◽  
Author(s):  
W. J. Bowen ◽  
K. Laki
Keyword(s):  
Binding Constant ◽  
Muscle Fibers ◽  
Component Analysis ◽  
Contractile Protein ◽  
Temperature Dependent ◽  
Molecular Rearrangement ◽  
Previous Conclusion ◽  
Apparent Binding Constant ◽  
Speed Of Shortening

Experiments reported here further support the previous conclusion that the shortening of glycerol-treated muscle caused by neutral Nessler's reagent (HgI2 in KI solution) is essentially due to the KI component. Analysis of the data obtained indicates that the apparent binding constant of KI to the contractile protein is 5 or 6 m/l. The same value for the binding ATP is about 10,000. The extent and speed of shortening in HgI2-KI solution are temperature dependent. The molecular rearrangement which results in the shortening of glycerol-treated fibers is considered to be directly related to the binding KI to the protein.

scholarly journals Raman optical activity study of poly-L-proline chains of various lengths

2010 ◽  
Vol 24 (3-4) ◽  
pp. 213-217 ◽  
Author(s):  
V. Profant ◽  
M. Šafarík ◽  
P. Bour ◽  
V. Baumruk
Keyword(s):  
Optical Activity ◽  
Conformational Stability ◽  
High Sensitivity ◽  
Wide Frequency Range ◽  
Peptide Chain ◽  
Helical Conformation ◽  
Polyproline Ii ◽  
Raman Optical Activity ◽  
Spectral Peaks ◽  
Chain Lengths

Raman and Raman optical activity (ROA) spectra of several oligo- and poly-L-proline samples of various chain lengths were measured in a wide frequency range between 120 and 1800 cm−1and analysed with respect to the main peptide chain conformation. Specifically, formation of polyproline II (PPII) helical conformation was studied in dependence on the increasing chain lengthNof the (L-proline)Nsample. Due to high sensitivity of the ROA technique to the conformational stability and rigidity of peptide chain we were able to determine the characteristic spectral peaks associated with formation of stable PPII helical conformation in studied systems. The most relevant peaks are located at 405, 535 and 945 cm−1. Additionally, based on our data analysis, we were able to determine the minimal length of (L-proline)Nchain necessary for creation of the stable PPII conformation asN= 6.

scholarly journals Application of DSC Method in Studies on Phase Transitions of Ni Superalloys

2017 ◽  
Vol 17 (4) ◽  
pp. 133-136 ◽  
Author(s):  
R. Przeliorz ◽  
J. Piątkowski
Keyword(s):  
Phase Transitions ◽  
Specific Heat ◽  
Protective Atmosphere ◽  
Scanning Calorimetry ◽  
Heating And Cooling ◽  
Complete Melting ◽  
Calorimetric Studies ◽  
Dsc Method ◽  
Dsc Curves

Abstract The paper presents results of calorimetric studies of foundry nickel superalloys: IN100, IN713C, Mar-M247 and ŻS6U. Particular attention was paid to determination of phase transitions temperatures during heating and cooling. The samples were heated to a temperature of 1500°C with a rate of 10°C⋅min-1 and then held at this temperature for 5 min. After a complete melting, the samples were cooled with the same rate. Argon with a purity of 99.99% constituted the protective atmosphere. The sample was placed in an alundum crucible with a capacity of 0.45 cm3. Temperature and heat calibration was carried out based on the melting point of high-purity Ni. The tests were carried out by the differential scanning calorimetry (DSC) using a Multi HTC high-temperature calorimeter from Setaram. Based on the DSC curves, the following temperatures were determined: solidus and liquidus, dissolution and precipitation of the γ’ phase, MC carbides and melting of the γ’/γ eutectic. In the temperature range of 100-1100°C, specific heat capacity of the investigated superalloys was determined. It was found that the IN713C and IN100 alloys exhibit a higher specific heat while compared to the Mar-M247 and ŻS6U alloys.

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