Indication Of Conformational Change Research Articles

Acetylcholine receptor: SH group reactivity as indicator of conformational changes and functional states.

Acetylcholine receptor: SH group reactivity as indicator of conformational changes and functional states.

Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP-pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers cah be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

The distance between two functionally significant regions of the 50 S Escherichia coli ribosome: the erythromycin binding site and proteins [formula omitted

The distance between the erythromycin binding site on the 50 S Escherichia coli ribosome and protein L7 has been measured by singlet-singlet energy transfer. A non-covalently bound erythromycin derivative, fluoroscein isothiocyanate erythromycylamine, was used as the acceptor. This derivative can be completely displaced from ribosomes by erythromycin, suggesting that they have the same binding site. 1,5-Iodoacetylethylenediamine naptholsulfonate-labeled protein L7 served as the fluorescent donor. It was reconstituted with salt/ethanol-washed 50 S cores. This readdition was accompanied by total recovery of elongation factor G-dependent GTPase activity. This suggests that the protein modification does not significantly perturb 50 S function or structure. Energy transfer measurements by both static and lifetime techniques were in good agreement. After consideration of various errors that enter the measurements and calculations, the L7-erythromycin distance is estimated to be 70 ± 10 Å. This long distance is interesting, since both sites may be involved in translocation. The fluorescent derivative of erythromycin was also used to study binding kinetics to the 50 S and 70 S ribosomes. Binding is a simple second-order step and proceeds about 11 times faster on the 70 S particle. Exchange of the fluorescent derivative with excess erythromycin is limited by the dissociation rate, and this is four times faster on the 70 S particle. These results suggest that the erythromycin site is more accessible on the 70 S particle, and may be an indication of conformational changes in the 50 S ribosome upon combination with the 30 S ribosome.

Reversible Conformational Changes of Myoglobin and Apomyoglobin

The “thermodynamic” hypothesis of protein conformation (1) states that the sequence of amino acids characteristic of a given protein is sufficient to determine its secondary and tertiary structure, for the molecule will assume the conformation thermodynamically most stable. In an analogous vein, the “conformation” hypothesis (2) proposes that the steric and noncovalent bonding properties of poly-cu-amino acid side chains dictate the tendency of these polymers to assume helical, extended, or random coil structures. These hypotheses need little alteration in order to include the possibly significant role of small cofactors and prosthetic groups. Such small molecules may interact with a protein in a specific fashion and thereby influence the relative stability of various folded states. Myoglobin and apomyoglobin offer an attractive system for evaluating the above hypotheses. A comparison of conformation-dependent parameters of the native and apoproteins will reveal what role, if any, is played by the heme moiety in the folding of myoglobin, while a study of reversible conformational changes of apomyoglobin will reflect the adequacy of the amino acid sequence in dictating the structure. The absence of sulfhydryl side chains or disulfide bridges in these proteins is particularly important. The elegant work of Anhnsen on the reversible unfolding of ribonuclease (3), which has placed the “thermodynamic” hypothesis on a secure experimental basis, depends on the oxidation of reduced disulfide bonds. It is thus not capable of distinguishing the role of the amino acid sequence in general from that of half-cystines in particular. The absence of this complicating factor in the myoglobin-apomyoglobin system therefore provides considerable simplification of interpretation. As an indication of conformational changes, we have used optical rotatory dispersion. Recent advances in our understanding of this technique as a measure of a-helical secondary structure (4-11) and recent improvements in instrumentation (12) have increased our confidence in the use and interpretations of ordinary rotatory dispersion. In this paper we report data which indicate (a) that a small loss in helix content, which is recovered on reconstitution, accompanies removal of the heme prosthetic group from myoglobin; (b) that the loss of helical structure of apomyoglobin that occurs on addition of urea can be completely reversed by dialysis against water or buffer; and (c) that apomyoglobin, restored in this manner to its native conformation, is capable of combining with heme to form myoglobin.