Pulsed-alkylation mass spectrometry for the study of protein folding and dynamics: development and application to the study of a folding/unfolding intermediate of bacterial luciferase.

A new method employing the classical techniques of chemical modification of proteins and the new technology of mass spectrometry, known as pulsed-alkylation mass spectrometry (PA/MS), has been developed to probe the dynamic structure of folding intermediates and folded complexes of proteins under a variety of conditions. This method is fast and simple, and the results are easily interpreted. PA/MS may provide an alternative to H/D exchange monitored either by NMR or by electrospray ionization mass spectrometry for some experiments; for others, it may provide access to questions not readily answered by available methods. The objective of PA/MS is to determine simultaneously the location and the extent of labeling of functional groups in a protein by measuring the reactivity of cysteines with N-ethylmaleimide, within the context of the conformation of the protein under specific conditions. The method can also be applied to chemical modification of other amino acid residues employing any of a vast array of reagents, depending upon the specifics of the protein under investigation. The enormous range of reactivity of the thiol groups of the cysteinyl residues in proteins and the change in reactivity upon denaturation or conformational rearrangement afford a large signal change that can be correlated with changes in accessibility of the thiol group. The information obtained from the correlation of observed thiol reactivity with the local environment of each cysteinyl residue in the structure of the folded protein can be supplemented by results obtained from fluorescence, circular dichroism, or other methods, to develop an understanding of the structure and dynamics of altered conformational states. With bacterial luciferase as a model system, we have applied PA/MS to investigate the structural differences between the native heterodimeric enzyme and a folding intermediate that is well-populated in 2 M urea. The thiol residues at positions 307, 324, and 325 of the alpha subunit were much more reactive with N-ethylmaleimide in the presence of 2 M urea than in the native enzyme, suggesting that the C-terminal region of the alpha subunit was less tightly packed in the folding intermediate. The apparent unfolding of the C-terminal region of the alpha subunit of the alphabeta structure in 2 M urea appears to mimic the unfolding of the C-terminal domain of the free alpha subunit, also in 2 M urea, described by Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145. The approach described here should be applicable to a wide array of problems that have in common the need to determine the locations of conformational changes in proteins. Application of PA/MS to the investigation of the relative thermodynamic stability of the coordination complexes of zinc within each of the six zinc-finger domains of MRE-binding transcription factor-1 (Zn(6) MTF-zf) in its free and DNA-bound forms is presented in the companion paper in this issue [Apuy, J. L., Chen, X., Russell, D. H., Baldwin, T. O., and Giedroc, D. P. (2001) Biochemistry 40, 15164-15175].

Protein contributions to redox potentials of homologous rubredoxins: an energy minimization study.

The energetic contributions of the protein to the redox potential in an iron-sulfur protein are studied via energy minimization, comparing homologous rubredoxins from Clostridium pasteurianum, Desulfovibrio gigas, Desulfovibrio vulgaris, and Pyrococcus furiosus. The reduction reaction was divided into 1) the change in the redox site charge without allowing the protein to respond and 2) the relaxation of the protein in response to the new charge state, focusing on the latter. The energy minimizations predict structural relaxation near the redox site that agrees well with that in crystal structures of oxidized and reduced P. furiosus rubredoxin, but underpredicts it far from the redox site. However, the relaxation energies from the energy-minimized structures agree well with those from the crystal structures, because the polar groups near the redox site are the main determinants and the charged groups are all located at the surface and thus are screened dielectrically. Relaxation energies are necessary for good agreement with experimentally observed differences in reduction energies between C. pasteurianum and the other three rubredoxins. Overall, the relaxation energy is large (over 500 mV) from both the energy-minimized and the crystal structures. In addition, the range in the relaxation energy for the different rubredoxins is large (300 mV), because even though the structural perturbations of the polar groups are small, they are very near the redox site. Thus the relaxation energy is an important factor to consider in reduction energetics.

Structural origins of redox potentials in Fe-S proteins: electrostatic potentials of crystal structures.

Redox potentials often differ dramatically for homologous proteins that have identical redox centers. For two types of iron-sulfur proteins, the rubredoxins and the high-potential iron-sulfur proteins (HiPIPs), no structural explanations for these differences have been found. We calculated the classical electrostatic potential at the redox site using static crystal structures of four rubredoxins and four HiPIPs to identify important structural determinants of their redox potentials. The contributions from just the backbone and polar side chains are shown to explain major features of the experimental redox potentials. For instance, in the rubredoxins, the presence of Val 44 versus Ala 44 causes a backbone shift that explains a approximately 50 mV lower redox potential in one of the four rubredoxins. This result is consistent with experimental redox potentials of five additional rubredoxins with known sequence. Also, we attribute the unusually lower redox potentials of two of the HiPIPs studied to a less positive electrostatic environment around their redox sites. Finally, molecular dynamics simulations of solvent around static rubredoxin crystal structures indicate that water alone is a major factor in dampening the contribution of charged side chains, in accord with experiments showing that mutations of surface charges produce relatively little effect on redox potentials.

The need for pediatric-specific triage criteria: results from the Florida Trauma Triage Study.

OBJECTIVE: The objective of the Florida Trauma Triage Study was to assess the performance of state-adopted field triage criteria. The study addressed three specific age groups: pediatric (age < 15 years), adult (age 15-54 years), and geriatric (age 55+ years). Since 1990, Florida has used a uniform set of eight triage criteria, known as the trauma scorecard, for triaging adult trauma patients to state-approved trauma centers. However, only five of the criteria are recommended for use with pediatric patients. This article presents the findings regarding the performance of the scorecard when applied to a pediatric population. DESIGN: We used state trauma registry data linked to state hospital discharge data in a retrospective analysis of trauma patients transported by prehospital providers to any acute care hospital within nine selected Florida counties between July 1, 1991, and December 31, 1991. We used cross-table and logistic regression analysis to determine the ability of triage criteria to correctly identify patients who were retrospectively defined as major trauma. We applied the field criteria to physiologic and anatomy/mechanism of injury data contained in the trauma registry to "score" the patient as major or minor trauma. To make our retrospective determination of major or minor trauma we used the protocols developed by an expert medical panel as described by E. J. MacKenzie et al. (1990). MAIN OUTCOME MEASURES: We calculated sensitivity, specificity, and the corresponding over- and undertriage rates by comparing patient classifications (major or minor trauma) produced by the triage criteria and the retrospective algorithm. We used logistic regression to identify which triage criteria were statistically significant in predicting major trauma. RESULTS: Pediatric cases accounted for 9.2% of the total study population, 6.0% of all hospitalized cases, and 6.8% of all trauma deaths. Of the 1505 pediatric cases available for analysis, the triage criteria classified 269 cases as expected major trauma and 1236 cases as expected minor trauma. The retrospective algorithm classified 78 cases as expected major trauma and 1427 cases as expected minor trauma. The resulting specificity is 84.8% (15.2% overtriage), and the sensitivity is 66.7% (33.3% undertriage). Logistic regression indicated that, of the eight state-adopted field triage criteria, only the Glasgow coma score, ejection from vehicle, and penetrating injuries have a statistically significant impact on predicting major trauma in pediatric patients. CONCLUSIONS: Although the state-adopted trauma scorecard, applied to a pediatric population, produced acceptable overtriage, it did not produce acceptable undertriage. However, our undertriage rate is comparable to the results of other published studies on pediatric trauma. As a result of the Florida Trauma Triage Study, a new pediatric triage instrument was developed. It is currently being field-tested.

Temperature dependence of the redox potential of rubredoxin from Pyrococcus furiosus: a molecular dynamics study.

Molecular dynamics simulations are used to evaluate the temperature dependent differences in structure, solvation, and energies for the iron-sulfur protein rubredoxin from the hyperthermophilic archebacterium Pyrococcus furiosus to understand the unusual temperature dependence of its redox potential [Adams, M. W. W. (1992) Adv. Inorg. Chem. 38, 341-396]. Simulations of both redox states performed at 295 and 363 K reveal that almost no backbone structure alteration occurs at the higher temperature and that the radius of gyration of the protein is temperature and redox state independent. The most striking change is that the penetration of the redox site by solvent molecules in the reduced from at 295 K, which was also seen in simulations of the reduced form of the mesophilic Clostridium pasteurianum rubredoxin at 295 K (Yelle, R. B., et al. (1995) Proteins 22, 154-167], is no longer seen to a significant extent in either redox state at 363 K. Comparing 295 to 363 K, the calculated change in the electrostatic potential of about -300 mV and in the negative of the potential energy of about -550 meV is consistent with the observed change in redox potential of -160 mV. Moreover, the calculated change is in the wrong direction if the penetrating water is excluded. These results show that changing solvent accessibility may be responsible for the temperature dependence of the redox potential of P. furiosus rubredoxin.

The failure of triage criteria to identify geriatric patients with trauma: results from the Florida Trauma Triage Study.

Since 1990, Florida has used a uniform set of eight triage criteria, known as the trauma scorecard, for triaging adult patients with trauma to state-approved trauma centers. If any one of the eight criteria are met, paramedics classify the patient as a "trauma alert" and transport to a state-approved trauma center. Widespread concern within the trauma care community that the scorecard was not providing an effective tool for adult trauma triage, particularly for older adults, was a motivating force for conducting an evaluation of the trauma scorecard's performance. Thus, the Florida Department of Health and Rehabilitative Services, Office of Emergency Medical Services initiated a research effort to assess the effectiveness of the state-adopted trauma triage criteria for adults, giving special attention to geriatric trauma. The results of the Florida Trauma Triage Study indicate that the eight triage criteria comprising the trauma scorecard produce unacceptable levels of undertriage in elderly patients (age 55 years or older) with life-threatening injuries.