Predicting changes in the status of patient-reported outcome measures after Birmingham Hip Resurfacing: an observational cohort study with a median follow-up of ten years.

AIMS: It is not known whether change in patient-reported outcome measures (PROMs) over time can be predicted by factors present at surgery, or early follow-up. The aim of this study was to identify factors associated with changes in PROM status between two-year evaluation and medium-term follow-up. PATIENTS AND METHODS: Patients undergoing Birmingham Hip Resurfacing completed the Veteran's Rand 36 (VR-36), modified Harris Hip Score (mHHS), Tegner Activity Score, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) at two years and a minimum of three years. A change in score was assessed against minimal clinically important difference (MCID) and patient-acceptable symptom state (PASS) thresholds. Binary logistic regression was used to assess the relationship between patient factors and deterioration in PASS status between follow-ups. RESULTS: Overall, 18% of patients reported reductions in mHHS total score exceeding MCID, and 21% reported similar reductions for WOMAC function scores. Nonetheless, almost all patients remained above PASS thresholds for WOMAC function (98%) and mHHS (93%). Overall, 66% of patients with mHHS scores < PASS at two years reported scores > PASS at latest follow-up. Conversely, 6% of patients deteriorated from > PASS to < PASS between follow-ups. Multivariable modelling indicated body mass index (BMI) > 27 kg/m2, VR-36 Physical Component Score (PCS) < 51, VR-36 Mental Component Score (MCS) > 55, mHHS < 84 at two years, female sex, and bone graft use predicted these deteriorating patients with 79% accuracy and an area under the curve (AUC) of 0.84. CONCLUSION: Due to largely acceptable results at a later follow-up, extensive monitoring of multiple PROMs is not recommended for Birmingham Hip Resurfacing patients unless they report borderline or unacceptable hip function at two years, are female, are overweight, or received a bone graft during surgery. Cite this article: Bone Joint J 2019;101-B:1431-1437.

The outcome and survival of metal-on-metal hip resurfacing in patients aged less than 50 years: a prospective observational cohort study with minimum ten-year follow-up.

AIMS: The aim of this study was to report the implant survival and patient-reported outcome measures (PROMs) in a consecutive series of patients aged less than 50 years at the time of arthroplasty using the Birmingham Hip Resurfacing system (BHR), with a minimum follow-up of ten years. PATIENTS AND METHODS: A total of 226 patients with osteoarthritis of the hip, who underwent BHR and presented to a single surgeon, were included in the study. Survival of the implant was confirmed by cross-checking with the Australian Orthopaedic Association National Joint Replacement Registry. Kaplan-Meier survival curves with 95% confidence intervals (CIs) were constructed. Pre- and postoperative PROMs were compared with t-tests, and postoperative scores were compared using anchor analysis with age and gender matched normative data. RESULTS: At median follow-up of 12 years (interquartile range (IQR) 10 to 13), six BHRs were revised, with a cumulative rate of survival of 96.8% (95% confidence interval (CI) 94.2 to 99.4) at 15 years, and with a significantly lower (p = 0.019) cumulative rate of revision than the national average for the same device at ten years. Most revisions (n = 4) were undertaken early, less than three years postoperatively, and occurred in women. Patient-reported general health (Veteran's Rand-36), disease state (Western Ontario and McMaster Universities Osteoarthritis Index), function (modified Harris Hip Score) and level of activity (Tegner activity score) maintained significant (p < 0.01 for each) improvements beyond ten years postoperatively and were equal to, or exceeded, age- and gender-matched normative data in more than 80% of the patients. CONCLUSION: Longer term PROMs after BHR, from a single surgeon, for patients aged less than 50 years remain under-reported. We found that the outcome after a BHR, at a minimum of ten years postoperatively, remained satisfactory, particularly for self-reported hip function.

Immunobullous disease and ulcerative colitis: a case series of six patients.

Cases of immunobullous skin disease associated with ulcerative colitis (UC) have been previously reported in the literature. There is no clear explanation for this association. In this series, we report six cases of immunobullous disease in patients with UC and discuss potential mechanisms of pathogenesis proposed to explain these concomitant diseases. The clinical presentation, immunopathology and treatment of six new cases are described and analysed. We report six patients, two with linear IgA bullous dermatosis (LABD), one with bullous pemphigoid (BP), one with mucous membrane pemphigoid (MMP) and two with IgA pemphigus. The patients' ages ranged from 33 to 66 years at the onset of their skin disease, and all but one case had a documented age of UC onset, confirmed with colonoscopy, prior to the development of skin disease. Direct immunofluorescence results in these patients demonstrated IgA basement membrane zone (BMZ) antibodies in the LABD cases, IgG antibodies at the BMZ in the BP and MMP cases, and IgA cell surface antibodies in the patients with IgA pemphigus. Additionally, indirect immunofluorescence was positive in one of the patients with LABD, the patient with BP and both of the patients with IgA pemphigus. The temporal association of UC and skin disease, in addition to the resolution of skin disease with total colectomy in one case, suggests colonic mucosal antigenic stimulation driving immune activation and leading to immunobullous skin disease.

Patient outcomes using Wii-enhanced rehabilitation after total knee replacement - the TKR-POWER study.

BACKGROUND: Home-based rehabilitation following total knee replacement surgery can be as effective as clinic-based or in-patient rehabilitation. The use of the Nintendo Wii has been postulated as a novel rehabilitation tool that adds an additional focus on balance and proprioception into the recovery protocol. The aim of the proposed clinical trial is to investigate the effectiveness of this novel rehabilitation tool, used at home for three months after total knee replacement surgery and to assess any lasting improvements in functional outcome at one year. METHODS/DESIGN: This will be a randomised controlled trial of 128 patients undergoing primary total knee replacement. The participants will be recruited preoperatively from three surgeons at a single centre. There will be no change to the usual care provided until 6 weeks after the operation. Then participants will be randomised to either the Wii-Fit group or usual rehabilitative care group. Outcomes will be assessed preoperatively, a 6-week post surgery baseline and then at 18 weeks, 6 months and 1 year. The primary outcome is the change in self-reported WOMAC total score from week 6 to 18 weeks. Secondary outcomes include objective measures of strength, function and satisfaction scores. DISCUSSION: The results of this clinical trial will be directly relevant for implementation into clinical practice. If beneficial, this affordable technology could be used by many patients to rehabilitate at home. Not only could it optimize the outcomes from their total knee replacement surgery but decrease the need for clinic-based or outpatient therapy for the majority. TRIAL REGISTRATION: (ACTRN12611000291987).

Can tibial coverage in total knee replacement be reliably evaluated with three-dimensional image-based digital templating?

OBJECTIVES: There remains a lack of data on the reliability of methods to estimate tibial coverage achieved during total knee replacement. In order to address this gap, the intra- and interobserver reliability of a three-dimensional (3D) digital templating method was assessed with one symmetric and one asymmetric prosthesis design. METHODS: A total of 120 template procedures were performed according to specific rotational and over-hang criteria by three observers at time zero and again two weeks later. Total and sub-region coverage were calculated and the reliability of the templating and measurement method was evaluated. RESULTS: Excellent intra- and interobserver reliability was observed for total coverage, when minimal component overhang (intraclass correlation coefficient (ICC) = 0.87) or no component overhang (ICC = 0.92) was permitted, regardless of rotational restrictions. CONCLUSIONS: Measurement of tibial coverage can be reliable using the templating method described even if the rotational axis selected still has a minor influence.

Different changes in slope between the medial and lateral tibial plateau after open-wedge high tibial osteotomy.

PURPOSE: In contrast to radiographic measurements, MRI provides multiple slices of the knee joint in the sagittal plane, making it possible to assess the medial and lateral tibial slope separately. The purpose of this study is to investigate the effect of medial open-wedge high tibial osteotomy (MOWHTO) on bony and meniscal slope in the medial and lateral tibiofemoral compartments. It was hypothesised that greater changes on the medial tibial plateau would be observed compared with the lateral one. METHODS: A retrospective analysis of prospectively collected data was performed on pre- and post-operative MRIs from 21 patients (17 men and 4 women; age 52 ± 9 years). Inclusion criteria were varus alignment, medial compartment osteoarthritis and election for a primary MOWHTO. Each patient had a preoperative and a post-operative high-resolution MRI (3Tesla, Magnetom Trio, Siemens AG) at an average follow-up of 2.1 years. A previously published method was used to measure bony and meniscal slope for each compartment. The difference between pre- and post-operative tibial slope for both compartments was calculated and associated with the amount of frontal correction. RESULTS: There was a significant increase in bony tibial slope in both compartments following MOWHTO. When a change in bony tibial slope was detected in an individual patient, the change was larger in the medial compartment, with the average change also significantly greater (p < 0.01) in the medial compartment (2.4° ± 1.3°) compared with the lateral compartment (0.9° ± 1.1°). There was also a significant increase (p < 0.01) in the lateral tibial meniscal slope of 0.9° ± 1.4°, which was equivalent to the change in the bony lateral slope. The amount of frontal correction was not significantly associated with the amount of change in slope. CONCLUSIONS: The results suggest that the modification of the bony slope is larger in the medial compartment after MOWHTO, which is likely related to the location of the hinge on the lateral tibial cortex. These findings suggest that consideration of the medial and lateral tibial slope intra-operatively could be important to identify the optimal location of the hinge. However, further studies are required before recommending any modification to the surgical technique, as the potential clinical consequences of tibial slope alterations remain unknown. LEVEL OF EVIDENCE: IV.

EPR-detected folding kinetics of externally located cysteine-directed spin-labeled mutants of iso-1-cytochrome c.

We report the application of our newly developed dielectric resonator-based flow and stopped-flow kinetic EPR systematically to probe protein folding in yeast iso-1-cytochrome c at cysteine-directed spin-labeled locations. The locations studied have not been previously directly probed by other techniques, and we observe them on a time scale stretching from 50 micros to seconds. On the basis of crystal structure and homology information, the following mutation-tolerant, externally located cysteine labeling sites were chosen (in helices, T8C, E66C, and N92C; in loops, E21C, V28C, H39C, D50C, and K79C), and labeling at these sites was not destabilizing. Dilution of denaturant was used to induce folding and thereby to cause a change in the spin label EPR signal as folding altered the motion of the spin label. Under folding conditions, including the presence of imidazole to eliminate kinetic trapping due to heme misligation, a phase of folding on the 20-30 ms time scale was found. This phase occurred not only at the T8C and N92C labeling sites in the N- and C-terminal helices, where such a phase has been associated with folding in these helices, but overall at labeling sites throughout the protein. In the absence of imidazole the 20-30 ms phase disappeared, and another phase having the time scale of 1 s appeared throughout the protein. There was evidence under all conditions for a burst phase on a scale of less than several milliseconds which occurred at labeling positions V28C, H39C, D50C, E66C, and K79C in the middle of the protein sequence. At spin-labeled D50C rapid-mix flow EPR indicated a very short approximately 50 micros phase possibly associated with the prefolding or compaction of the loop to which D50 belongs. Spin labels have been criticized as perturbing the phenomena which they measure, but our spin labeling strategy has reported common kinetic themes and not perturbed, disconnected kinetic events.

Variable velocity liquid flow EPR applied to submillisecond protein folding.

We have developed a variable velocity, rapid-mix, continuous-flow method for observing and delineating kinetics by dielectric resonator-based electron paramagnetic resonance (EPR). The technology opens a new facet for kinetic study of radicals in liquid at submillisecond time resolution. The EPR system (after Sienkiewicz, A., K. Qu, and C. P. Scholes. 1994. Rev. Sci. Instrum. 65:68-74) accommodated a miniature quartz capillary mixer with an approximately 0.5 microliter delivery volume to the midpoint of the EPR-active zone. The flow velocity was varied in a preprogrammed manner, giving a minimum delivery time of approximately 150 microseconds. The mixing was efficient, and we constructed kinetics in the 0.15-2. 1-ms time range by plotting the continuous wave EPR signal taken during flow versus the reciprocal of flow velocity. We followed the refolding kinetics of iso-1-cytochrome c spin-labeled at Cysteine 102. At 20 degrees C, upon dilution of guanidinium hydrochloride denaturant, a fast phase of refolding was resolved with an exponential time constant of 0.12 ms, which was consistent with the "burst" phase observed by optically detected flow techniques. At 7 degrees C the kinetic refolding time of this phase increased to 0.5 ms.

Q-band ENDOR (electron nuclear double resonance) of the high-affinity ubisemiquinone center in cytochrome bo3 from Escherichia coli.

Electron nuclear double resonance (ENDOR) was performed on the protein-bound, stabilized, high-affinity ubisemiquinone radical, QH*-, of bo3 quinol oxidase to determine its electronic spin distribution and to probe its interaction with its surroundings. Until this present work, such ENDOR studies of protein-stabilized ubisemiquinone centers have only been done on photosynthetic reaction centers whose function is to reduce a ubiquinol pool. In contrast, QH*- serves to oxidize a ubiquinol pool in the course of electron transfer from the ubiquinol pool to the oxygen-consuming center of terminal bo3 oxidase. As documented by large hyperfine couplings (>10 MHz) to nonexchangeable protons on the QH*- ubisemiquinone ring, we provide evidence for an electronic distribution on QH*- that is different from that of the semiquinones of reaction centers. Since the ubisemiquinone itself is physically nearly identical in both QH*- and the bacterial photosynthetic reaction centers, this electronic difference is evidently a function of the local protein environment. Interaction of QH*- with this local protein environment was explicitly shown by exchangeable deuteron ENDOR that implied hydrogen bonding to the quinone and by weak proton hyperfine couplings to the local protein matrix.

Dielectric resonator-based side-access probe for muscle fiber EPR study.

We present a novel dielectric resonator (DR)-based resonant structure that accommodates aqueous sample capillaries in orientations that are either parallel (i.e., side-access) or perpendicular to the direction of an external (Zeeman) magnetic field, B(0). The resonant structure consists of two commercially available X-band DRs that are separated by a Rexolite spacer and resonate in the fundamental TE(01delta) mode. The separator between the DRs is used to tune the resonator to the desired frequency and, by appropriately drilled sample holes, to provide access for longitudinal samples, notably capillaries containing oriented, spin-labeled muscle fibers. In contrast to the topologically similar cylindrical TE(011) cavity, the DR-based structure has distinct microwave properties that favor its use for parallel orientation of lossy aqueous samples. For perpendicular orientation of a dilute (6.25 microM) aqueous solution of IASL spin label, the S/N ratio was at least one order of magnitude better for the side-access DR-based structure than for a standard TE(102) cavity. EPR spectra acquired for maleimide spin-labeled myosin filaments also revealed ca. 10 times better S/N ratio than those obtained with a standard TE(102) cavity. For the side-access DR with sample capillaries oriented either parallel or perpendicular to the external magnetic field, the Q- and filling factors are in good agreement with the theoretical estimates derived from the distribution of magnetic (H(1)) and electric (E(1)) components.

Methadone titration in opioid-resistant cancer pain.

AIM: To assess the use of methadone in patients with cancer pain who fail to respond to increasing doses of other opioids or experience intolerable side-effects from them. METHOD: Inpatients of a specialist palliative care unit were titrated onto oral methadone. The dose was calculated as 10% of the previous morphine equivalent dose, up to maximum of 40 mg, given every 3 h as required for analgesia. When daily requirements were stable it was divided into two regular doses. Pain was assessed on a five-point verbal rating score (VRS): a good response was defined as a fall in VRS of two points or more. Results are expressed as median (range). RESULTS: Thirty-three patients (13 men, 20 women, age 61 (34-91) years), 26 with inadequate analgesia and seven with intolerable opioid related side-effects, were converted to methadone from diamorphine (12), morphine (19) or fentanyl (two). Morphine equivalent dose was 480 (20-1200) mg/day prior to titration. Pain was neuropathic (11), nociceptive (three) or mixed (19). Stabilisation on methadone was complete in 3 (2-18) days in 29 (88%) patients at 80 (20-360) mg/day. Twenty-six (78%) had a good response. Four (12%) patients were withdrawn during titration (three entered terminal phase, one failed to respond). During follow-up 15 (45%) required alteration of methadone dose. Twenty-three (70%) patients were discharged home at 12 (4-26) days. In all cases the stable dose of methadone was less than the previous morphine equivalent, and there was a weak correlation between them. CONCLUSIONS: This method of methadone titration often results in improved pain control in patients with morphine resistance or intolerance. It requires careful titration in a specialist inpatient unit as there is no reliable formula for dose equivalence.

Dielectric resonator-based flow and stopped-flow EPR with rapid field scanning: A methodology for increasing kinetic information.

We report methodology which combines recently developed dielectric resonator-based, rapid-mix, stopped-flow EPR (appropriate for small, aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic field where the scanning is preprogrammed to occur at selected times after the start of flow. This methodology gave spectroscopic information complementary to that obtained by stopped-flow EPR at single fields, and with low reactant usage, it yielded more graphic insight into the time evolution of radical and spin-labeled species. We first used the ascorbyl radical as a test system where rapid scans triggered after flow was stopped provided "snapshots" of simultaneously evolving and interacting radical species. We monitored ascorbyl radical populations either as brought on by biologically damaging peroxynitrite oxidant or as chemically and kinetically interacting with a spectroscopically overlapping nitroxide radical. In a different biophysical application, where a spin-label lineshape reflected rapidly changing molecular dynamics of folding spin-labeled protein, rapid scan spectra were taken during flow with different flow rates and correspondingly different times after the mixing-induced inception of protein folding. This flow/rapid scan method is a means for monitoring early immobilization of the spin probe in the course of the folding process.

Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3.

We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.

Electronic structural information from Q-band ENDOR on the type 1 and type 2 copper liganding environment in wild-type and mutant forms of copper-containing nitrite reductase.

Q-band ENDOR elucidated proton and nitrogen hyperfine features to provide spin density information at ligands of blue-green Type 1 and catalytic Type 2 copper centers in nitrite reductase. The blue-green Type 1 center of nitrite reductase has a redox, electron-transfer role, and compared to the blue center of plastocyanin, it has the following structural differences: a shortened Cu-Smet bond length, a longer Cu-Scys bond length, and altered ligand-copper-ligand bond angles (Adman, E. T., Godden, J. W., and Turley, S. (1995) J. Biol. Chem. 270, 27458-27474). The hyperfine couplings of the two Type 1 histidine (N delta) ligands showed a larger percentage difference from each other in electron spin density than previously reported for other blue Type 1 proteins, while the cysteine beta-proton hyperfine couplings, a measure of unpaired p pi spin density on the liganding cysteine sulfur, showed a smaller electron spin density. A mutation of the Type 1 center, M182T, having the copper-liganding Met182 transformed to Thr182, caused the center to revert to an optically "blue" center, raised its redox potential by approximately 100 mV, and led to the loss of activity (prior paper). Surprisingly, in M182T there was no change from native Type 1 copper either in the histidine or cysteine hyperfine couplings or in g values and Cu nuclear hyperfine couplings. The conclusion is that the optical and redox alterations due to changed Type 1 methionine ligation need not be concurrent with electron spin delocalization changes in the HOMO as reported from its essential cysteine and histidines. A detailed picture of the nitrogen couplings from the three histidine (N epsilon) ligands of the Type 2 center indicated a substantial ( approximately 200%) electronic hyperfine inequivalence of one of the histidine nitrogens from the other two within the Type 2 HOMO and thus provided evidence for electronic distortion of the Type 2 site. In the presence of the nitrite substrate, hyperfine couplings of all histidines diminished. We suggest that this nitrite-induced decreased covalency would correlate with an increased Type 2 redox potential to assist electron transfer to the Type 2 center. Dipole-coupled, angle-selected exchangeable proton features, observed over a range of g values, predicted a ligand-water proton distance of 2.80 A from copper, and these water protons were eliminated by nitrite. His287 is not a Type 2 ligand but is positioned to perturb an axial water or a nitrite of Type 2 copper. In the presence of nitrite the mutant H287E showed no evidence for the loss of water protons and no diminished ligand histidine covalency. H287E has vastly diminished activity (prior paper), and the ENDOR information is that NO2- does not bind to Type 2 copper of H287E. In summary, the electronic information from this study of native and suitably chosen mutants provided a test of the highest occupied molecular orbital (HOMO) wave function at Type 1 and Type 2 coppers and an intimate electronic insight into functional enzymatic properties.

Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1. Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102.

The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68-74], gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds. The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins. The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochrome c at the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype. This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme [Mchaourab, H. S. Lietzow, M. A., Hideg, K., & Hubbell, W. L. (1996) Biochemistry 35, 7692-7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P., & Millhauser, G. L. (1991) Biochemistry 30, 5515-5523]. The C-terminal cytochrome c helix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding. Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein. We observed an approximately 50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M. The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5. The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0. At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV-Vis that indicated a slower folding where heme misligation may be involved. Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction. The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics. The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted. The motion of the attached nitroxide in the folded protein appears to be restricted about the carbon and sulfur bonds which tether it to the cysteine. The original point of cysteine sulfur attachment is approximately 11 A from the heme iron within the C-terminal helix near its interface with the N-terminal helix, but the low-temperature EPR spin probe line width showed that the probe lies more distant (> 15 A) from the heme iron. By all physical evidence, the protein labeled at cysteine102 folded, but the spin probe in this prototype system perturbed packing which lowered the thermal melting temperature, the free energy of folding, the guanidinium concentration at the midpoint of the unfolding transition, the m parameter of the denaturant, and the helical CD signature. This study prepares the way for study of protein folding/unfolding kinetics using EPR spectroscopy of spin-labels placed at specific cysteine-mutated sites within

Double-stacked dielectric resonator for sensitive EPR measurements.

A new approximate method for predicting the resonant frequencies and for solving the field distribution problem of a cylindrical dielectric resonator (DR) is developed. The model proposed in this paper bridges the gap between rigorous and accurate finite-element or Green function-based numerical methods on the one hand and on the other hand, simple approximate solutions in which the field distribution can be described analytically, but the resulting frequency is accurate within a few percent only. In the method described here, the approximate solution for the microwave field distribution is modified by substituting different values of the radial separation constants inside and outside of the diskshaped DR. The model is generalized for the double-stacked DR structure and enables one to introduce corrections that take into account the presence of the shielding walls and of the cylindrical sample hole. Good agreement is found between experimental and calculated results for both the single and double-stacked structures that are designed around commercially available X-band DRs (9-10 GHz). For the resonant frequency of the lowest transverse-electric TEzero1 delta mode that is commonly used for EPR measurements, the accuracy of the method is better than 1%. Experimentally measured resonator filling factors are also in good agreement with those theoretically estimated. Both the theory and the experimental results suggest that the double-stacked DR structure with finite spacing between the ceramic cylinders is the most suitable for EPR measurements of long lossy samples.

Endonuclease III interactions with DNA substrates. 1. Binding and footprinting studies with oligonucleotides containing a reduced apyrimidinic site.

The binding of endonuclease III from Escherichia coli to damaged DNA has been studied using gel shift and footprinting assays. Oligonucleotides containing a reduced apyrimidinic (AP) site were used since reduction of the AP site blocks the beta-elimination reaction catalyzed by the enzyme and yields a noncleavable substrate. The Kobs for a 13-mer carrying a centrally located reduced AP site is (2 x 10(6)-(2 x 10(7) M-1, while the Kobs for a 13-mer with no damage is (4.5 x 10(3)-(3.2 x 10(4) M-1 (approximately a 500-fold difference). Larger oligonucleotides would not enter a gel when endonuclease III was bound so that binding constants to oligonucleotides longer than 13 base pairs could not be determined directly. Competition assays suggest that the Kobs measured for both damaged and undamaged 13-mers is a minimum value and that the Kobs for larger oligonucleotides could be an order of magnitude greater. Fluorescence quenching on related 19-mers yielded a specific binding constant for the 19-mer carrying a centrally located reduced AP site for 4 x 10(7) M-1 and a nonspecific binding constant to an undamaged 19-mer of approximately 10(5) M-1 [Xing, D., Dorr, R., Cunningham, R. P., & Scholes, C. P. (1995) Biochemistry 34, 2537-2544]. Several footprinting reagents were used to determine the size and location of the endonuclease III binding site on damaged oligonucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Endonuclease III interactions with DNA substrates. 2. The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching.

We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs. Endonuclease III has two tryptophans, Trp132 in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron-sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O'Handley, S. F., & Cunningham, R. P. (1992) Science 258, 434-440]. The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment. Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are differently accessible to anionic quencher. Significant (approximately 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weight duplex undamaged poly(dAdT). The apparent second-order nonspecific binding constant to poly(dAdT) was 4 x 10(7) M-1, and there were approximately 12 base pairs per endonuclease III binding site for binding to poly(dAdT). This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA. A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was approximately 400-fold larger than to the undamaged 19-mer.(ABSTRACT TRUNCATED AT 250 WORDS)