The complete denaturation and subsequent renaturation and reconstitution of a polytopic integral membrane protein are demonstrated. Delipidated bacteriorhodopsin (Huang, K.-S., Bayley, H., and Khorana, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 323-327) is completely denatured when transferred into 88% formic acid or anhydrous trifluoroacetic acid as shown by NMR and circular dichroism sp...

We have studied nucleic acid double helix destabilization mediated by purified calf helix-unwinding proteins, measuring ultraviolet hyperchromicity to detect helix melting. Both,calf unwinding protein 1 (UPl) and a high salt eluting protein fraction are found to depress strongly the helix melting temperature (T,) of the synthetic alternating copolymers poly[d(AT) ] and poly[r(AU)], indicating t...

Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consu...

It has been shown in an earlier paper from this laboratory (1) that a univalent active fragment, obtained from rabbit antibody directed against bovine serum albumin, can spontaneously regain its original physical properties, and most of its original biological activity, after being fully unfolded. The unfolding agent used was concentrated guanidine hydrochloride. Several independent measurement...

alpha-Crystallin, a major lens protein, has many of the properties of a molecular chaperone, but its ability to assist refolding of proteins has been less certain. In the present work it was shown that alpha-crystallin specifically increased the reactivation of guanidine-denatured glyceraldehyde-3-phosphate dehydrogenase with most of the activity being recovered. In the incubation mixture the r...

Figure A Overview of Steps Involved in a Cot Analysis. (A) Nuclear DNA is sheared into 200-500 bp fragments by high-speed blending, and (B) fragment size is checked by gel electrophoresis. (C) Sheared DNA is precipitated, dissolved in 0.03, 0.12, or 0.5 M sodium phosphate buffer (SPB), and aliquots are loaded/sealed into glass microcapillary tubes/ampoules. (D) One tube/ampoule is (E) placed in...

Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incubation at 4 °C, while heat-treated γ-glutamyltranspeptidase of Bacillus subtilis did not. Fluorescent spectra, CD spectra, and native polyacrylamide gel electrophoresis analysis suggested that the dimer of E. coli γ-glutamyltranspeptidase was separated into protomers by heat-treatment, but was renat...