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Biomedical subjects

J M Sturtevant

Publications and source records attributed to J M Sturtevant.

At least 19 recordsLinked to original sources
PubMed1992

A differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In continuation of our earlier work on the effects of amino acid replacements on the thermodynamics of the thermal unfolding of T4 lysozyme [Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, 3788-3792; Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1991) Biochemistry 30, 1887-1891; Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1992) Biochemistry 31, 1643-1647], we report here a study by differential scanning calorimetry of the effects of five replacements at Ile3. Four of these replacements, those with Glu, Phe, Pro, and Thr, caused apparent destabilizations, while the replacement by Leu led to a small apparent stabilization. The largest observed destabilization (Ile3Pro) amounted to -3.0 kcal mol-1 in free energy at pH 2.00 and 38.8 degrees C (the denaturational temperature of the wild-type protein at this pH), and the largest stabilization amounted to +1.2 kcal mol-1 at pH 3.00 and 53.6 degrees C.

Bacteriophage T4↗
PubMed1992

Thermodynamic measurements of the contributions of helix-connecting loops and of retinal to the stability of bacteriorhodopsin.

Thermodynamic studies of bacteriorhodopsin (BR) have been undertaken in order to investigate the factors that stabilize the structure of a membrane protein. The stability of the native, intact protein was compared to that of protein with retinal removed, and/or cleaved in one or two of the loops connecting the transmembrane helices. The stability was assessed using differential scanning calorimetry and thermal denaturation curves obtained from ultraviolet circular dichroism and absorption spectroscopy. Retinal binding and the loop connections were each found to make a small contribution to stability, and even a sample that was cleaved twice as well as bleached to remove retinal denatured well above room temperature. Removal of retinal destabilized the protein more than cleaving once, and about as much as cleaving twice. Retinal binding and the connections in the loops were found to stabilize BR in independent ways. Cleavage of the molecule into fragments did not reduce the intermolecular cooperativity of the denaturation. Dilution of the protein by addition of excess lipid in order to eliminate the purple membrane crystal lattice also did not alter the cooperativity. These results are used to compare the relative importance of various contributors to the stability of BR.

Bacteriorhodopsins↗
PubMed1992

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.

Calorimetry, Differential Scanning↗
PubMed1979

Thermodynamics of the denaturation of lysozyme in alcohol--water mixtures.

The thermal denaturation of lysozyme was studied at pH 2 in aqueous mixtures of methanol, ethanol, and 1-propanol by high sensitivity differential scanning calorimetry (DSC). The most obvious effect of alcohols was the lowering of Td, the temperature of denaturation, increasingly with higher alcohol concentration and longer alkyl chain. Both the calorimetric and van't Hoff enthalpies of denaturation initially increased and then decreased with increasing alcohol concentration, the ratio of the two enthalpies being nearly unity, 1.007 +/- 0.011, indicating the validity of the two-state approximation for the unfolding of lysozyme in these solvent systems. The reversibility of the denaturation was demonstrated by the reversibility of the DSC curves and the complete recovery of enzymic activity on cooling. The changes in heat capacity on unfolding decreased with increasing alcohol concentration for each alcohol. Experimentally determined values of denaturation temperature and of entropy and heat capacity changes were used to derive the additional thermodynamic parameters delta G degrees and delta S degrees for denaturation as a function of temperature for each alcohol--water mixture. Comparison of the thermodynamic parameters with those reported [Pfeil, W., & Privalov, P.L. (1976) Biophys. Chem. 4, 23--50] in aqueous solution at various values of pH and guanidine hydrochloride concentration showed that these latter changes have no effect on the heat capacity changes, whereas the addition of alcohols causes a sharp decrease.

Alcohols↗
PubMed1979

The lambda repressor contains two domains.

Papain digestion of the lambda phage repressor produces two fragments that are relatively resistant to further digestion. One includes the amino terminus (residues 1-92) and the other the carboxyl terminus (residues 132-236). Calorimetry shows that the amino-terminal fragment denatures near 50 degrees C and that the carboxyl-terminal fragment denatures near 70 degrees C. Intact repressor undergoes two denaturations, one near 50 degrees C and another near 70 degrees C. These and other data show that lambda repressor consists of two domains joined by a "connector" 40 amino acids long that is sensitive to proteases. The amino-terminal domain binds DNA, and the carboxyl-terminal domain oligomerizes.

Amino Acid Sequence↗
PubMed1979

Thermotropic behavior of some fluorodimyristoylphosphatidylcholines.

The thermotropic behavior of three fluorinated phospholipids has been studied by means of high-sensitivity differential scanning calorimetry. The three lipids are 1,2-di(X,X-difluoromyristovyl)phosphatidylcholine with X = 4,8, or 12. The compound with X = 4 has a higher transition temperature than dimyristoylphosphatidylcholine whereas the other two have lower values. All three lipids have transition enthalpies approximately twice that of the unsubstituted lipid. It was found the substituted lipids form strikingly nonideal mixtures with the parent compound. In view of these observations it is evident that results obtained using these or related compounds as probes of bilayer or membrane behavior must be interpreted with due caution. Some of the properties exhibited by geminal fluorine atoms in fluorinated phospholipids appear to be similar to those of cis double bonds in lipids.

Calorimetry↗
PubMed1978

Phase behavior of lipids from Halobacterium halobium.

Mixtures of dipalmitoylphosphatidylcholine with purple membrane lipids, red membrane lipids, or total lipids of Halobacterium halobium have been studied with differential scanning calorimetry. A comparison of red and purple membrane lipids reveals no difference in their phase behavior, indicating that lipid phase behavior plays no role in the in vivo separation of red and purple membranes. The effects of variation of the salt content of the suspending solution have also been examined. Studies of the melting behavior of these mixtures as H. halobium lipid content is varied suggest that the gel to liquid-crystal transition does not occur in the lipids of H. halobium.

Bacteriorhodopsins↗
PubMed1978

High-sensitivity scanning calorimetric study of mixtures of cholesterol with dimyristoyl- and dipalmitoylphosphatidylcholines.

A highly sensitive and stable scanning microcalorimeter is employed in a reinvestigation of the effect of cholesterol on multilamellar suspensions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). Below 20 mol % cholesterol the DPPC mixtures give heat-capacity curves each of which can be resolved into a narrow and a broad peak, suggesting the coexistence of two immiscible solid phases; above 20 mol % only the broad peak is observed and this disappears at about 50 mol %. The DMPC mixtures show a more complicated behavior; from about 13.5 to 20 mol % cholesterol the observed curves appear to be the sum of three component peaks. As with the DPPC mixtures, only a single broad peak is observed above 20 mol % cholesterol, and this broad peak becomes undetectable above about 50 mol %. These results are discussed.

Calorimetry, Differential Scanning↗
PubMed1978

Phase transitions of the purple membranes of Halobacterium halobium.

Purple membranes of Halobacterium halobium were studied by differential scanning calorimetry. No transition was detected at temperatures below 70 degrees C. A small endothermic transition was seen at about 80 degrees C and a larger one at 100 degrees C. The larger transition is the irreversible denaturation of bacteriorhodopsin. The smaller transition is accompanied by a change in the visible absorption spectrum and is believed to be reversible, involving a cooperative change in crystalline structure of the membrane.

Bacteriorhodopsins↗
PubMed1978

Nature of the gel to liquid crystal transition of synthetic phosphatidylcholines.

High sensitivity differential scanning calorimetry is employed in the study of the gel to liquid crystal phase transition of a highly purified sample of dipalmitoylphosphatidylcholine in multilamellar bilayer suspension. It is concluded from the calorimetric data that the purity of the sample is better than 99.94 mol % and that the transition closely approximates an isothermal first-order transition.

Calorimetry↗
PubMed1978

Proposed temperature-dependent conformational transition in D-amino acid oxidase: a differential scanning microcalorimetric study.

A number of authors have reported observations on D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3.] that they have interpreted in terms of a temperature-dependent conformational transition having a van't Hoff enthalpy amounting to more than 1 cal per g of protein (1 cal = 4.184J). No indication of this transition is obtained by using a differential scanning calorimeter having a sensitivity considerably in excess of that required to detect such a transition. The implications of this discrepancy are discussed.

Apoenzymes↗
PubMed1977

Studies of the lipid phase transitions of Escherichia coli by high sensitivity differential scanning calorimetry.

High sensitivity adiabatic differential scanning calorimetry was performed on lipids, membrane vesicles, and whole cells of Escherichia coli enriched in particular unsaturated fatty acids by genetic means. Information concerning the shape of the transition is discussed. Transitions with an asymmetric shape reminiscient of a second order transition were observed. Comparison between the lipid transition observed in whole cells, membrane vesicles, and extracted lipids enriched in elaidate reveal some basic similarities. Studies of synthetic lipids were undertaken in an attempt to interpret the shapes of these transitions as a function of the lipid components of the membrane.

Calorimetry↗
PubMed1977

A calorimetric examination of stable and fusing lipid bilayer vesicles.

Mixed lipid samples containing dimyristoylglycerophosphocholine and small amounts of myristic acid were examined calorimetrically. Examination of multilamellar and small vesicle samples indicated that upon heating small vesicles combine to form more extended structures. An exothermic peak (at 19 . 5 degrees C) can be associated with the structural transformation. The enthalpy for this process, which may be interpreted as vesicle-vesicle fusion, is found to be approx.--2 kcal/mol.

Calorimetry↗
PubMed1977

Incorporation of saturated fatty acids into phosphatidylcholine bilayers.

The transition temperature of dipalmitoylglycerophosphocholine in multi-lamellar aqueous suspensions, as observed by high-sensitivity differential scanning calorimetry, is raised from 41.4 to 61.5 degrees C by addition of palmitic acid at a mole fraction of 0.67. It appears that the fatty acid chains pack in the hexagonal lattice with the lipid chains in a one-to one ratio, thereby eliminating the destabilizing crowding of the phosphatidylcholine head groups. A similar effect on dilauroylglycerophosphocholine is produced by lauric acid. The stabilizing effect is not produced in full measure by acids of different chain lengths, nor by alcohols or saturated hydrocarbons of the same chain length.

Gels↗
PubMed1977

Energetics of the cooperative and noncooperative binding of nicotinamide adenine dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase at pH 6.5 and pH 8.5. Equilibrium and calorimetric analysis over a range of temperature.

The binding of nicotinamide adenine dinucleotide (NAD+) to yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) has been studied at pH 6.5 and 8.5, at 5,25, and 40 degrees C, by calorimetry, fluorometry, spectrophotometry, equilibrium dialysis, and flow dialysis. As reported earlier for pH 7.3 (Velick S.F., Baggott, J.P., and Sturtevant, J.M. (1971), Biochemistry 10, 779), the binding is accompanied by enthalpy changes which become rapidly more negative as the temperature increases, with delta Cp = -500 to -750 cal deg-1 (mole of NAD+ bound)-1, and by entropy changes which also, as required by the large negative delta Cp, become rapidly more negative with increasing temperature. The binding data at pH 6.5 can be fitted on the basis of either four identical noninteracting sites, or of four sites showing a small degree of negative cooperativity. The data at pH 8.5, particularly at 40 degrees C, require the introduction of positive cooperativity, as was previously shown by Kirschner et al. (Kirschner, K., Eigen, M., Bittman, R., and Voigt, B. (1966), Proc. Natl. Acad. Sci. U.S.A. 56, 1661), and can be equally well fitted on the basis of a sequential model (Adair, G.S. (1925), J. Biol. Chem. 63, 529) or a concerted model (Monod, J., Wyman, J., and Changeux, J.P. (1965), J. Mol. Biol. 12, 88). It is proposed that the observed thermodynamic changes are largely the result of a hydrophobic effect due to a decrease in the exposure of nonpolar groups to the solvent, and of a tightening of the protein structure when the coenzyme is bound with concomitant decrease in the number of easily excitable internal degrees of freedom.

Binding Sites↗
PubMed1977

A calorimetric investigation of single stranded base stacking in the ribo-oligonucleotide A7.

Differential scanning calorimetry has been employed to determine the energy change associated with single stranded base stacking in the ribo-oligonucleotide A7. A total enthalpy change of 20.3 kcal (mole of heptamer)-1 was measured. This corresponds to 2.9 kcal (mole of adenine)-1 or 3.4 kcal (mole of A-A stack)-1 if one assumes that all six stacking interactions are energetically equivalent. These results represent the first direct determination of this important parameter for a ribo-oligonucleotide. It is noted that the calorimetrically determined value reported here is considerably lower than any of the previously published van't Hoff enthalpies but is consistent with values that can be derived from other calorimetric data.

Adenine Nucleotides↗