Hydrogen-tritium exchange by apoferritin and ferritin

The out-exchange kinetics of tritium from apoferritin, ferritin of various iron contents, and apoferritin subunits were examined. The exchange kinetics indicated no detectable conformational differences in the tetracosamer with and without hydrous ferric oxide in the internal cavity of the molecule. The data for apoferritin subunits were markedly different from those for the tetracosameric state. The exchange kinetics for apoferritin were consistent with a rapid exchange of water between the internal cavity of the protein and the bulk solvent outside the protein shell.     

Hydrogen-tritium exchange survey of allosteric effects in hemoglobin

The oxy and deoxy forms of hemoglobin display major differences in H-exchange behavior. Hydrogen-tritium exchange experiments on hemoglobin were performed in the low-resolution mode to observe the dependence of these differences on pH (Bohr effect), organic phosphates, and salt. Unlike a prior report, increasing pH was found to decrease the oxy-deoxy difference monotonically, in general accordance with the alkaline Bohr effect. A prior report that the H-exchange difference between oxy- and deoxyhemoglobin vanishes at pH 9, and thus appears to reflect the Bohr effect alone, was found to be due to the borate buffer used, which at high pH tends to abolish the oxy-deoxy difference in a limited region of the H-exchange curve. Effects on hemoglobin H exchange due to organic phosphates parallel the differential binding of these agents (inositol hexaphosphate more than diphosphoglycerate, deoxy more than oxy, at low pH more than at high pH). Added salt slows H exchange of deoxyhemoglobin and has no effect on the oxy form. These results display the sensitivity of simple H-exchange measurements for finding and characterizing effects on structure and dynamics that may occur anywhere in the protein and help to define conditions for higher resolution approaches that can localize the changes observed.     

The random walk description for isotope exchange in a polypeptide

Isotope exchange in a polypeptide is considered from the point of view in which the boundary point between helix and coil regions of a polypeptide behaves like a weakly asymmetric random walker. We assume that the boundary point is reflected completely at the ends of a polypeptide. The equilibrium fraction of helix region is obtained under this assumption, and this is also confirmed by computer simulation. The experimental results of isotope exchange can be explained in this situation. On the other hand. the rate constant of exchange of a residue given by experiments can also be explained by another assumption, as considered before (M. Fujiwara and N. Saitô, Polym. J. 9 (1977) 625.), in which the nucleations of coil states take place in the helix region. Which of the two is of major importance is left to further studies.     

Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation

Interactions of eukaryotic 5-dimethylaminonaphthalene-1-sulfonyl-initiation factor 2 (eIF-2) from rabbit reticulocytes and the guanine nucleotide exchange factor ( GEF ), Met-tRNAf, GTP, and GDP were monitored by changes in fluorescence anisotropy and radioactive filtration assays. At 1 mM Mg2+, radioactive filtration assays demonstrate that GEF is necessary for nucleotide exchange. We did not observe a GDP dependence in the association reaction of eIF-2 X GEF for GDP concentrations from 0.01 to 20 microM. This is in disagreement with the model: eIF-2 X GDP + GEF in equilibrium eIF-2 X GEF + GDP. The addition of GTP caused a decrease in fluorescence anisotropy which is interpreted as a dissociation of eIF-2 X GEF . We propose an asymmetrical model of ternary complex (eIF-2 X GTP X Met-tRNAf) formation where 1) GDP does not displace GEF and 2) GTP replaces GEF and presumably GDP. For reticulocyte eIF-2, phosphorylation of the alpha subunit greatly inhibits protein synthesis. This inhibition derives neither from failure of GEF to bind to eIF-2(alpha P) nor from greatly enhanced binding of GEF . The inhibition results from the requirement of very high levels of GTP (100 microM) to dissociate the eIF-2(alpha P) X GEF complex.     

Monte Carlo generation of polypeptide random coil conformations; the persistence vector and the chain vector distribution

Polypeptide random coil conformations of various chain lenghts (N = 5, 10, 20, 40, 80 peptide units) are generated by a Monte Carlo procedure. The characteristic ratio obtained for the sets of generated conformations is identical with the exact value calculated with the average transformation matrix procedure, indicating the equivalence of the two treatments. On the basic of the generated sets of conformations the length and direction of the persistence vector (the averaged chain vector expressed in the reference frame of the first two skeletal bonds) are investigated for various chain lengths. The radial distribution function for the chain vector shows the length of the chain vector for small polypeptides (N = 5, 10) not to deviate far from its most probable value. Also for larger chains up to chains of 80 peptide units very significant deviations from a gaussian distribution are observed.The distribution of the length of the vector connecting the remote end of the chain with the end of the persistence vector exhibited behavior much doser to the gaussian approximation, an improvement especially significant for the short chains.     

Acid proteases from species of Mucor. IV. Hydrogen-tritium exchange of Mucor miehei protease.

The kinetics of hydrogen-tritium exchange were studied in the range pH-3 for both the fully and partially tritiated protein. Exchange constants for an intermediate class and slow class of hydrogens were determined and found to give a parabolic curve characteristic of acid and base catalysis about the observed pHmin of 4.03. The anomalous rate retardation on the acid portion of the curve was attributed to electrostatic interactions which could be evaluated quantitatively from the titration data. Partial tritation and pH cross-over experiments indicated that the rank order was pH-independent thus eliminating the possiblitity of a major conformational change. Consequently, the data are most likely explicable in terms of restricted solvent accessibility.     

Phosphorylation and guanine nucleotide exchange on polypeptide chain initiation factor-2 from Artemia embryos

Eukaryotic initiation factor-2 (eIF-2) from Artemia embryos is able to exchange guanine nucleotides at the same rate in the presence or absence of Mg2+ when the reaction is carried out with either purified eIF-2 at 30 degrees C or less purified preparations at any temperature (10-30 degrees C). No exchange factor appears to catalyze this reaction. However, with purified eIF-2 at lower temperatures (10 degrees C) the exchange is clearly impaired by Mg2+ and this impairment is overcome by the guanine nucleotide exchange factor (GEF) of rabbit reticulocytes. Thus, Artemia eIF-2 is able to exchange guanine nucleotides by two alternative mechanisms that may reflect two states of the protein. Phosphorylation of the eIF-2 alpha subunit by the heme-controlled inhibitor (HCI) of rabbit reticulocytes abolishes the GEF-dependent reaction, but has no effect on the factor-independent one. The search for eIF-2 alpha kinases in Artemia embryo led to the detection of only one such enzyme, which was identified as a casein kinase type II. None of the exchange reactions is affected by the phosphorylation of the eIF-2 alpha subunit by this kinase, suggesting that, irrespective of the kind of mechanism for guanine nucleotide exchange that is actually operating in Artemia, it might not be a target for regulation by eIF-2 alpha phosphorylation.     

Mechanism of the nucleotide exchange reaction in eukaryotic polypeptide chain initiation. Characterization of the guanine nucleotide exchange factor as a GTP-binding protein

Polypeptide chain initiation in mammalian systems is regulated at the level of the guanine nucleotide exchange factor (GEF). This multisubunit protein catalyzes the exchange of GDP bound to eukaryotic initiation factor 2 (eIF-2) for GTP. Although various models have been proposed for its mode of action, the exact sequence of events involved in nucleotide exchange is still uncertain. We have studied this reaction by three different experimental techniques: (a) membrane filtration assays to measure the release of [3H]GDP from the eIF-2.[3H]GDP binary complex, (b) changes in the steady-state polarization of fluorescamine-GDP during the nucleotide exchange reaction, and (c) sucrose gradient analysis of the total reaction. The results obtained do not support the reaction as written: eIF-2.GDP + GEF in equilibrium eIF-2.GEF + GDP. The addition of GEF alone does not result in the displacement of eIF-2-bound GDP. The release of bound GDP is dependent on the presence of both GTP and GEF, and this argues against the possibility of a substituted enzyme (ping-pong) mechanism for the guanine nucleotide exchange reaction. An important finding of the present study is the observation that GTP binds to GEF. The Kd value of 4 microM for GTP was estimated (a) by the extent of quenching of tryptophan fluorescence of GEF in the presence of GTP and (b) by the binding of [3H]GTP to GEF as measured on nitrocellulose membranes. The GEF-dependent release of eIF-2-bound GDP was studied at several constant concentrations of one substrate (GTP or eIF-2.GDP) while varying the second substrate concentration, and the results were then plotted according to the Lineweaver-Burk method. Taken together, the results of GTP and eIF-2.GDP binding to GEF and the pattern of the double-reciprocal plots strongly suggest that the guanine nucleotide exchange reaction follows a sequential mechanism.     

Hydrogen-tritium exchange kinetics of soybean trypsin inhibitor (Kunitz). Solvent accessibility in the folded conformation.

The hydrogen exchange kinetics of Kunitz soybean trypsin inhibitor (STI) has been studied at pH 2, 3, and 6.5. From the temperature dependence of proton exchange at low pH, THE CONTRIBUTION OF MAJOR, REVERSIBLE PROTEIN UNFOLDING To the hydrogen exchange kinetics has been determined. Exchange directly from the folded conformation is characterized by an apparent activation energy (E*app) of approximately 25 kcal/mol, close to that of the chemical exchange step. At pH 6.5 the protein is more temperature stable than at low pH, and exchange of all but congruent to 8 protons can be observed to exchange with E*app congruent to 27 kcal/mol. This implies that all but congruent to 8 protons are accessible to exchange with solvent in the solution structure of folded STI. Estimates can be made of the average number of water molecules per molecule of STI consistent with a solvent accessibility model of hydrogen exchange kinetics. These estimates indicate that very few water molecules within the protein matrix are necessary to explain the exchange data. Calculations are done for the STI hydrogen exchange kinetics at pH 3, 30 degrees, approximating STI structure by a sphere of radius = 18 A. These calculations indicate an average of congruent to 4 water molecules in the shell from 13 to 16 A. from the center of the molecule, while less than 1 water molecule is indicated in the innermost 13 A. These calculations also suggest that there are congruent to 190 water molecules associated with the outermost 1.5-2 A of the sphere. While these values are consistent with a hydrophobic region in the central protein matrix, they indicate more solvent accessibility in the outer 1/3 of the molecule than the static accessibility estimates made from X-ray coordinates. Our results suggest that any protein movements or fluctuations responsible for solvent accessibility in proton exchange processes are localized in the outer regions of the globular structure.     

Hydrogen-tritium exchange titration of the histidine residues in bovine heart cytochrome c and analysis of their microenvironment.

Microenvironments of the three histidine residues located at the positions 18, 26, and 33 from the amino terminus in bovine heart cytochrome c were analysed in solution by the hydrogen-tritium exchange titration method, which has been developed in this laboratory. Histidine-18, which is liganded to the heme iron, and histidine-26 did not incorporate tritium in native state, indicating that the two are located in solvent inaccessible hydrophobic regions. Histidine-33 was labeled with tritium to an appreciable extent and seemed to be partially buried in the molecule. The pKa value estimated for histidine-33 was 6.1 at 37 degrees by the tritium exchange titration, suggesting that the residue interacts very weakly with a neighboring cationic group. These results seem to be compatible with the tertiary structure of the protein deduced from the X-ray crystallographic analysis.     

Polymerization mechanism of polypeptide chain aggregation

The misfolding of polypeptide chains and aggregation into the insoluble inclusion body state is a serious problem for biotechnology and biomedical research. Developing a rational strategy to control aggregation requires understanding the mechanism of polymerization. We investigated the in vitro aggregation of P22 tailspike polypeptide chains by classical light scattering, nondenaturing gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis (PAGE), and computer simulations. The aggregation of polypeptide chains during refolding occurred by multimeric polymerization, in which two multimers of any size could associate to form a larger aggregate and did not require a sequential addition of monomeric subunits. The cluster-cluster polymerization mechanism of aggregation is an important determinant in the kinetic competition between productive folding and inclusion body formation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 333-343, 1997.