Determinants of backbone dynamics in native BPTI: cooperative influence of the 14-38 disulfide and the Tyr35 side-chain.

15Nitrogen relaxation experiments were used to characterize the backbone dynamics of two modified forms of bovine pancreatic trypsin inhibitor (BPTI). In one form, the disulfide between Cys14 and Cys38 in the wild-type protein was selectively reduced and methylated to generate an analog of the final intermediate in the disulfide-coupled folding pathway. The second form was generated by similarly modifying a mutant protein in which Tyr35 was replaced with Gly (Y35G). For both selectively reduced proteins, the overall conformation of native BPTI was retained, and the relaxation data for these proteins were compared with those obtained previously with the native wild-type and Y35G proteins. Removing the disulfide from either protein had only small effects on the observed longitudinal relaxation rates (R1) or heteronuclear cross relaxation rates (nuclear Overhauser effect), suggesting that the 14-38 disulfide has little influence on the fast (ps to ns) backbone dynamics of either protein. In the wild-type protein, the pattern of residues undergoing slower (micros to ms) internal motions, reflected in unusually large transverse relaxation rates (R2), was also largely unaffected by the removal of this disulfide. It thus appears that the large R2 rates previously observed in native wild-type protein are not a direct consequence of isomerization of the 14-38 disulfide. In contrast with the wild-type protein, reducing the disulfide in Y35G BPTI significantly decreased the number of backbone amides displaying large R2 rates. In addition, the frequencies of the backbone motions in the modified protein, estimated from R2 values measured at multiple refocusing delays, appear to span a wider range than those seen in native Y35G BPTI. Together, these observations suggest that the slow internal motions in Y35G BPTI are more independent in the absence of the 14-38 disulfide and that formation of this bond may lead to a substantial loss of conformational entropy. These effects may account for the previous observation that the Y35G substitution greatly destabilizes the disulfide. The results also demonstrate that the disulfide and the buried side-chain influence the dynamics of the folded protein in a highly cooperative fashion, with the effects of removing either being much greater in the absence of the other.

Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI.

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 --> Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 micros time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.

Mutations of iso-1-cytochrome c at positions 13 and 90. Separate effects on physical and functional properties.

Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c. The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three-dimensional structures are available. We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso-1-cytochrome c; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein. The mutated proteins [R13C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys and Cys102-->Thr mutations), [D90C, C102T]cytochrome c (iso-1-cytochrome c containing Asp90-->Cys and Cys102-->Thr mutations) and [R13C, D90C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys, Asp90-->Cys, and Cys102-->Thr mutations) are functional in vivo. Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups. The stability of mutant [R13C, C102T]cytochrome c resembles that of [C102T] cytochrome c, whereas the stability of [D90C, C102T]cytochrome c resembles the stability of [R13C, D90C, C102T]cytochrome c. The activity of cytochrome-c oxidase using cytochrome c was monitored polarographically. Compared to the wild-type or [C102T]cytochrome c, which shows two kinetic phases with cytochrome-c oxidase, [D90C, C102T]cytochrome c has much the same profile; [R13C, C102T]cytochrome c and [R13C, D90C, C102T]cytochrome c exhibit one kinetic phase with decreased activity. Electron-transfer activity of the mutant cytochromes c is inhibited by Hg2+. The inhibition is highest for the triple mutant, less for [R13C, C102T]cytochrome c, even less for [D90C, C102T]cytochrome c and insignificant for the wild type. It would appear as though the stability of the triple mutant follows the changes that result from the Asp90-->Cys mutation while the activity changes follow those of the Arg13-->Cys mutation.

Strategies for pooling data in occupational epidemiological studies.

In occupational epidemiology, most of the populations at risk are of limited size, and therefore pooling of the experience of different groups with similar exposure is desirable. We present and compare five different strategies for pooling of such data, including the "default" strategy of present reporting practices, which have the disadvantage that the experience of many small units is not recorded because of the low statistical power of small populations. A simple summation strategy including cases and controls from diverse groups, while attractively simple, may be fallacious when cases and controls have different proportions which are confounded with the risk ratios. Separate summation of observed and expected numbers of cases avoids the risk of fallacy, but may be unduly influenced by the results in one large plant. A "Clinical Trial" type of strategy in which all of the data are collected by a common protocol and therefore considered as a single data set is administratively complex and does not seem well suited for occupational epidemiology. Probability pooling seems to have attractive features, avoiding most of the disadvantages of the other strategies, and having the added feature that weighting for exposure and duration of follow-up can be included. Probability pooling requires exact probability statements for each study. Examples are given for pooling of data from exposures of operating room personnel to anaesthetic agents, cohorts of workers exposed to asbestos, and cohorts of workers exposed to man-made mineral fibres. A working group is proposed to recommend procedures and to assist in interpreting pooling activities.

Emergency room admissions, meteorologic variables, and air pollutants: a path analysis.

Daily hospital emergency room admissions at hospitals located within 8 km of Los Angeles Basin monitoring stations at Long Beach, Lennox, Azusa, and Riverside, California, were examined for correlations with pollutant and meteorologic variables for 1974-1975. By conventional correlation and regression with lagged and temporospatial analysis, the authors could not distinguish effects of pollution by particulate sulfate from those due to meteorologic variables and oxidant. The authors use a variety of structural models and path analysis to estimate "direct" effects on emergency room admissions of maximum temperature, humidity, wind velocity, barometric pressure, carbon monoxide, sulfur dioxide, nitric oxide, high-volume suspended particulates, coefficient of haze, nitrogen dioxide, oxidant, and sulfate. Criteria for choice of models included plausibility of pairwise dependence relationships, magnitude of the correlations with emergency room admissions, and examination of the partial correlation matrix. Their results show that a variety of models gave similarly large path coefficients for a given location for the following variables: maximum temperature at each site; sulfate at Long Beach and Lennox but not at Riverside which nevertheless had the highest sulfate means; oxidant at Azusa which had the highest oxidant levels. At other locations, despite substantial and significantly elevated correlation coefficients, oxidant had a small or negative path coefficient. After considering other possible factors, the authors conclude that sulfate pollution at Lennox and Long Beach had an important and possibly causal association with demand for emergency room admissions. This demonstrates the usefulness of using a variety of structural models in the analysis of ecologic data.