Ribonuclease temperature

Zale, S. E. and Klibanov, A. M., Why does ribonuclease irreversibly inactivate at high temperature , Biochemistry, 25, 5432, 1986. 

Fig. 12. Thermal denaturation for ribonuclease Tj as followed by VCD, from 20° to 65°C. The matrix descriptors determined for the native state and the unfolded high-temperature data are indicated. The values indicate a loss of the helix segment but maintenance of sheet segments. Also listed are the spectrally determined fractional contributions (FC) to the secondary structure. When combined with the segment analysis, this implies that the residual sheet segments must be very short. Reprinted with permission from Pancoska, P., et al. (1996). Biochemistry 35(40), 13094-13106, the American Chemical Society.

Qi et al. (1998) have demonstrated that ribonuclease A exhibits behavior like that of cytochrome c. The burst phase observed on dilution of Gdm HCl-denatured RNase A is mimicked exactly by reduced RNase A. The latter, when carboxamidomethylated to prevent oxidation, has a CD at 222 nm that is nearly independent of temperature and indicative of extensive unfolding at zero denaturant. 

Figure 4.8 Cation-exchange liquid chromatography of basic proteins. Column, Asahipak ES502C eluent, 20 min linear gradient of sodium chloride from 0 to 500 mM in 50 mM sodium phosphate buffer pH 7.0 flow rate, 1 ml min-1 temperature, 30 °C detection, UV 280 nm. Peaks 1, myoglobin from horse skeletal muscle (Mr 17 500, pi 6.8-7.3) 2, ribonuclease from bovine pancreas (Mr 13 700, pi 9.5-9.6) 3, a-chymotrypsinogen A from bovine pancreas (Mr 257 000, pi 9.5) and 4, lysozyme from egg white (Mr 14 300, pi 11.0-11.4). (Reproduced by permission from Asahikasei data)...

Figure 2.11. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for tryptophan in a model medium (glycerol) at different temperatures (a) and singletryptophan proteins (b). 1, Whiting parvalbumin, pH 6.S in the presence of Ca2+ ions 2, ribonuclease Th pH 6.5 3, ribonuclease C2, pH 6.5 4, human serum albumin, pH 7.0, +10"4 M sodium dodecyl sulfate 5, human serum albumin, pH 3.2 6, melittin, pH 7.5, +0.15 M NaCl 7, protease inhibitor IT-AJ from Actinomyces janthinus, pH 2.9 8, human serum albumin, pH 7.0 9, -casein, pH 7.5 10, protease inhibitor IT-AJ, pH 7.0 11, basic myelin protein, pH 7.0 12, melittin in water. The dashed line is the absorption spectrum of tryptophan.

Differences between the spectra of fluorescence and phosphorescence are immediately obvious. For all tryptophans in proteins the phosphorescence spectrum, even at room temperature, is structured, while the fluorescence emission is not. (Even at low temperatures the fluorescence emission spectrum is usually not structured. The notable exceptions include a-amylase and aldolase, 26 protease, azurin 27,28 and ribonuclease 7, staphylococcal endonuclease, elastase, tobacco mosaic virus coat protein, and Drosophila alcohol dehydrogenase 12. )... 

One simple case of disordered structure involves many of the long charged side chains exposed to solvent, particularly lysines. For example, 16 of the 19 lysines in myoglobin are listed as uncertain past C8 and 5 of them for all atoms past C/J (Watson, 1969) for ribonuclease S Wyckoff et al. (1970) report 6 of the 10 lysine side chains in zero electron density in trypsin the ends of 9 of the 13 lysines refined to the maximum allowed temperature factor of 40 (R. Stroud and J. Chambers, personal communication) and in rubredoxin refined at 1.2 A resolution the average temperature factor for the last 4 atoms in the side chain is 9.2 for one of the four lysines versus 43.6, 74.4, and 79.3 for the others. Figure 57 shows the refined electron density for the well-ordered lysine and for the best of the disordered ones in ru-... 

An explanation for these observances in the cases of ethanol and PEG may arise from hydrophobic interactions (i.e., methylene groups, methyl) with the unfolded state of the protein at elevated temperatures. This idea is supported by studies of the interaction of several alkylureas (methyl-, ACVdimethyl-, ethyl-, and butylureas) with the thermal unfolding of ribonuclease A, where it was shown... 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Marsh et al. (1983) have shown that SFV can still infect BHK-21 cells. Moreover, the release of nucleocapsid into the cytoplasm (as measured by the ribonuclease assay) proceeds, albeit 40% more slowly than at 37 C. Thin-section electron microscopy confirms that SFV particles are not transferred to lysosomes at this temperature. 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

Despite considerable biochemical work, high-resolution crystal structure determination of native RNase A and S, and some medium-resolution studies of RNase A-inhibitor complexes, a number of questions existed concerning the details of the catalytic mechanism and the role of specific amino acids. Study of the low-temperature kinetics and three-dimensional structures of the significant steps of the ribonuclease reaction was designed to address the following questions. 

The preceding summary and Fig. 20 present a frame-by-frame account of the pathway for ribonuclease catalysis, based predominandy on knowledge of the structures of the various intermediates and transition states involved. The ability to carry out such a study is dependent on three critical features (1) crystals of the enzyme which diffract sufficiently well to permit structural resolution to at least 2 A (2) compatibility of the enzyme, its crystals, and its catalytic kinetic parameters with cryoenzymology so as to permit the accumulation and stabilization of enzyme-substrate complexes and intermediates at subzero temperatures in fluid cryosolvents with crystalline enzyme and (3) the availability of suitable transition state analogs to mimic the actual transition states which are, of course, inaccessible due to their very short lifetimes. The results from this investigation demonstrate that this approach is feasible and can provide unparalleled information about an enzyme at work. 

The ultimate objective of an X-ray cryoenzymological study is the mapping of the structures of all kinetically significant species along the reaction pathway. In the case of ribonuclease A this has been largely achieved, as described above. Other enzymatic reactions now await application of the same techniques. Unfortunately, not all crystalline enzymes lend themselves to study by this method. In some cases it may be impossible to find a suitable cryoprotective mother liquor in others, the reaction may occur too rapidly at ordinary temperature. A reaction with Acat of 10 seconds and an activation enthalpy of —6 kcal mol will not be quenched even at — 75°C. The approach we have described in this article can be applied to only a small number of enzymes. Two likely candidates for successors to ribonuclease are the enzymes yeast triosephosphate isomerase and porcine pancreatic elastase. 

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