Domain separation and characterization of PriC, a replication restart primosome factor in Escherichia coli.

In Escherichia coli the oriC-independent primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex by DNA damage. Primosome is thought to form via two pathways: one PriA dependent and the other PriA independent. PriC is a key protein in the replication restart of the PriA-independent pathway. In this study, we determined that PriC was divided into two domains. Then, we obtained information that: (i) the C-terminal domain preferentially binds to single-stranded DNA (ssDNA); (ii) the binding of PriC to ssDNA depends on salt concentration; and (iii) the binding site size of PriC is approximately 7-9 nucleotides. The protease digestion of PriC suggested that a possible DNA-binding site is the N-terminus of the C-terminal domain where basic amino acid residues are concentrated. Interestingly, α-helical induction of the C-terminal domain of PriC occurred after the addition of DNAs. Also, we examined the role of heptad repeat of leucine or valine residues in the C-terminal domain and PriC oligomerization. This study describes the structure and function analysis of PriC which forms the primosome complex in replication restart.

Structural characteristic of folding/unfolding intermediate of pokeweed anti-viral protein revealed by time-resolved fluorescence.

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.

A distinct binding mode of archaeal ribonuclease P proteins to RNA.

The ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshii comprises RNA (PhopRNA) and five proteins. We analyzed the RNA binding mode of the protein, using a pair of complementary fluorescence-labeled oligoribonucleotides. Fluorescence resonance energy transfer (FRET)-based assays suggested that the RNase P proteins assist PhopRNA in attaining a functionally active conformation via a distinct mode of binding.

The structural mechanism of the inhibition of archaeal RelE toxin by its cognate RelB antitoxin.

The archaeal toxin, aRelE, in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 inhibits protein synthesis, whereas its cognate antitoxin, aRelB, neutralizes aRelE activity by forming a non-toxic complex, aRelB-aRelE. The structural mechanism whereby aRelB neutralizes aRelE activity was examined by biochemical and biophysical analyses. Overexpression of aRelB with an aRelE mutant (ΔC6), in which the C-terminal residues critical for aRelE activity were deleted, in Escherichia coli allowed a stable complex, aRelB-ΔC6, to be purified. Isothermal titration of aRelE or ΔC6 with aRelB indicated that the association constant (Ka) of wild-type aRelB-aRelE is similar to that of aRelB-ΔC6, demonstrating that aRelB makes little contact with the C-terminal active site of aRelE. Overexpression of deletion mutants of aRelB with aRelE indicated that either the N-terminal (pos. 1-27) or C-terminal (pos. 50-67) fragment of aRelB is sufficient to counteract the toxicity of aRelE in E. coli cells and the second α-helix (α2) in aRelB plays a critical role in forming a stable complex with aRelE. The present results demonstrate that aRelB, as expected from its X-ray structure, precludes aRelE from entering the ribosome, wrapping around the molecular surface of aRelE.

The quenching-resolved fluorescence spectrum and its application to studies of the folding/unfolding of trypsin inhibitor from seeds of the bitter gourd.

With reference to the local conformation of a protein, it is interesting to differentiate the individual fluorescence properties of included tryptophan residues without modification. The fluorescence spectrum of bitter gourd trypsin inhibitor (BGTI) was separated into two emission bands by the quenching-resolved fluorescence method. One emission band was given as a fraction with the Stern-Volmer quenching constant, 44.9 x 10(-3) M(-1), against the fluorescence quenching by KI, and it showed an emission maximum intensity at 341 nm. The fluorescence quenching constant of the other band was 1.58 x 10(-3) M(-1), and the maximum wavelength was found at 337 nm. These separated emissions were due to the fluorescence of Trp54 and Trp9 of BGTI. The quenching resolved-fluorescence spectrum was effectively applied to the precise description of the polar circumstances surrounding the Trp residues in the unfolding intermediate state of BGTI. The results suggested that the molten globule-like state of BGTI adopted such a peculiar conformation that the helix domain including Trp9 was packed more densely while the other loop domain partially unfolded.

Steady-state and time-resolved fluorescence spectroscopic studies on interaction of the N-terminal region with the hairpin loop of the phytocystatin Scb.

The steady-state and time-resolved fluorescence spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure.

Fluorescence decay characteristics of indole compounds revealed by time-resolved area-normalized emission spectroscopy.

Time-resolved fluorescence spectroscopy of tryptophan residue has been extensively applied to the studies on structure-function relationships of protein. Regardless of this, the fluorescence decay mechanism and kinetics of tryptophan residue in many proteins still remains unclear. Previous studies have demonstrated that conformational heterogeneity and relaxation dynamics are both involved in the peculiar multiexponential decay kinetics in subnanosecond resolution. In the present study, we characterized the fluorescence decay property of six indole compounds in glycerol by resolving the contribution of conformational heterogeneity and relaxation dynamics. We applied the time-resolved area-normalized fluorescence emission spectrum (TRANES) method for the fluorescence decay analysis. The results of TRANES, time-dependent shift of fluorescence spectral center of gravity, and fluorescence decay simulation demonstrated that the dielectric relaxation process independent of intrinsic rotamer/conformer and the individual fluorescence lifetime gives the peculiarity to the fluorescence decay of indole compounds. These results confirmed that TRANES and time-dependent spectral shift analysis are potent methods to resolve the origin of multiexponential decay kinetics of tryptophyl fluorescence in protein.

Protein-protein interaction on lysozyme crystallization revealed by rotational diffusion analysis.

Intermolecular interactions between protein molecules diffusing in various environments underlie many biological processes as well as control protein crystallization, which is a crucial step in x-ray protein structure determinations. Protein interactions were investigated through protein rotational diffusion analysis. First, it was confirmed that tetragonal lysozyme crystals containing fluorescein-tagged lysozyme were successfully formed with the same morphology as that of native protein. Using this nondisruptive fluorescent tracer system, we characterized the effects of sodium chloride and ammonium sulfate concentrations on lysozyme-lysozyme interactions by steady-state and time-resolved fluorescence anisotropy measurements and the introduction of a novel interaction parameter, k(rot). The results suggested that the specific attractive interaction, which was reflected in the retardation of the protein rotational diffusion, was induced depending on the salt type and its concentration. The change in the attractive interactions also correlated with the crystallization/precipitation behavior of lysozyme. Moreover, we discuss the validity of our rotational diffusion analysis through comparison with the osmotic second virial coefficient, B(22), previously reported for lysozyme and those estimated from k(rot).

The unfolding of alpha-momorcharin proceeds through the compact folded intermediate.

The unfolding of alpha-momorcharin was systematically investigated using steady-state and time-resolved tryptophan fluorescence, circular dichroism and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. These spectroscopic studies demonstrated that alpha-momorcharin unfolded through a compact folded intermediate state. The content of alpha-helix was increased, Trp192 approached closer to the side of active site and its rotational motion was restricted by being equilibrated with 2-3 M of guanidine hydrochloride. Furthermore, the binding of ANS with alpha-momorcharin was more suppressed to show that the hydrophobic parts would not be accessed to the protein surface but rather be sealed off in this specific conformation state. These results suggest that the structure of alpha-momorcharin holds the more compact conformation as an incipient state for unfolding, which is the sharp contrast to beta-momorcharin that gives the characteristics of the generally known molten globule state.

Heterogeneous packing in the folding/unfolding intermediate state of bitter gourd trypsin inhibitor.

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2-3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.

Plan reproducibility of intraoperatively custom-built linked seeds compared to loose seeds for prostate brachytherapy.

PURPOSE: Few studies have compared the implant quality of linked and loose seeds for prostate brachytherapy. This study aimed to evaluate and compare plan reproducibility of intraoperatively built custom linked seeds and loose seeds for prostate brachytherapy. MATERIAL AND METHODS: Between December 2010 and March 2014, 76 localized prostate cancer patients received Iodine-125 brachytherapy with external beam radiotherapy. Linked and loose seeds were implanted in 39 and 37 patients, respectively. The primary endpoint was the mean (± standard deviation) of the absolute change in the minimum dose received by 90% of the prostate volume between intraoperative and post-operative planning (ΔD90) to confirm plan reproducibility. Comparisons between the groups were evaluated using 2-sample t tests. RESULTS: The ΔD90 values were 6.95 ± 11.6% and -0.41 ± 8.5% for the loose and linked seed groups, respectively (p < 0.01). The linked seed group showed decreased post-operative D90 (118.8% vs. 127.2%), V150 (51.7% vs. 66.7%), and RV100 (0.44 ml vs. 0.61 ml) compared to the loose seed group (p < 0.01), whereas lung migration tended to be reduced (0% vs. 8%). CONCLUSIONS: The plan reproducibility of the linked seed group was better than that of the loose seed group. Moreover, the linked seed group showed less migration and lower rectal dose.

Prostate-specific antigen nadir within 12 months as an early surrogate marker of biochemical failure and distant metastasis after low-dose-rate brachytherapy or external beam radiotherapy for localized prostate cancer.

Prostate-specific antigen nadir (nPSA) after radiotherapy for localized prostate cancer has been investigated as a predictor. However, nPSA usually requires several years, limiting its clinical utility. We investigated the significance of nPSA within 12 months (nPSA12) after low-dose-rate prostate brachytherapy (LDR-PB) or external beam radiotherapy (EBRT) on treatment outcomes. Between 2006 and 2014, 663 patients with prostate cancer were treated with LDR-PB or EBRT at two institutions. Four hundred and seventy-four men received LDR-PB and 189 men received EBRT, without androgen deprivation therapy. The Kaplan-Meier method was used for biochemical failure (BF)-free survival (BFFS) and distant metastasis (DM)-free survival (DMFS) analyses, and multivariable Cox regression analysis was performed. The median follow-up was 61.3 months. The median nPSA12 in the LDR-PB and EBRT cohorts was 0.7 and 1.0 ng/mL, respectively. The 7-year BFFS and DMFS rates in LDR-PB patients with nPSA12 ≤ 0.7 ng/mL were 99.1% and 99.5%, respectively; when nPSA12 was >0.7 ng/mL, they were 90.2% and 94.8%, respectively. In EBRT patients with nPSA12 ≤ 1.0 ng/mL, BFFS and DMFS rates were 85.4% and 98.5%, respectively; when nPSA12 was >1.0 ng/mL, they were 67.1% and 87.2%, respectively. nPSA12 was an independent predictor of BF and DM in both cohorts (LDR-PB, P = 0.004 and 0.020, respectively; EBRT, P = 0.005 and 0.041, respectively). The nPSA12 after LDR-PB or EBRT is significantly associated with treatment outcomes of prostate cancer. Higher nPSA12 may identify patients at high risk of relapse who might benefit from salvage treatment.

Spectrally and time-resolved fluorescence spectroscopic study on melittin-calmodulin interaction.

The origin of multi-exponential fluorescence decay property of tryptophan (Trp) in protein has been in controversy, and dielectric relaxation is thought to be one of the most plausible candidates of that origin. In this study, we studied melittin-calmodulin interaction on the concept of dielectric relaxation by spectrally and time-resolved fluorescence spectroscopy. Trp residue in melittin demonstrated drastic change in its dielectric relaxation rate and scale by binding with calmodulin. Expected change of relaxation rate suggested that dielectric relaxation accounts for multi-exponential property of fluorescence decay. We also examined the time variation of radiative and non-radiative decay rates. That result demonstrated the distinct difference profiles of non-radiative decay rate of Trp in melittin and the complex.

The partially unfolded state of beta-momorcharin characterized with steady-state and time-resolved fluorescence studies.

The specific conformation of partially unfolded state of beta-momorcharin was characterized through the steady-state and time-resolved fluorescence spectroscopic studies on a single Trp-190 which located adjacently to the active site. The content of secondary structure was retained, the binding of ANS was remarkably enhanced, and the correlation time of entire protein rotation was prolonged at the partially unfolded state formed by being equilibrated with the mild concentration of guanidine hydrochloride. The time-resolved fluorescence depolarization and excitation energy transfer analysis suggest that Trp-190 approached 2 A closer to Tyr-70 and was hidden from the exposure to the protein surface, while the rotational correlation time and freedom of its segmental motion were shortened and enhanced, respectively. These results suggest that the transient folding/unfolding intermediate state of beta-momorcharin adopt the specific conformation at the vicinity of the active site, although it exhibits very similar properties with those of the generally known molten-globule state.

Quantitative analysis of genitourinary toxicity after iodine-125 brachytherapy for localized prostate cancer: Followup of the International Prostate Symptom Score and Overactive Bladder Symptom Score.

PURPOSE: To analyze genitourinary toxicity by followup of the International Prostate Symptom Score (IPSS) and Overactive Bladder Symptom Score (OABSS) after prostate brachytherapy. METHODS AND MATERIALS: Six hundred eighty patients were treated with iodine-125 brachytherapy for localized prostate cancer. IPSS, OABSS, and two categories of IPSS questions (storage symptom score [IPSS-S] and voiding symptom score [IPSS-V]) were evaluated. RESULTS: The median followup was 54 months (range, 24-108). All scales showed rapid increases followed by gradual decreases. The median times to IPSS peak and resolution were 1 and 6 months, respectively. The resolution rates of IPSS, IPSS-S, IPSS-V, and OABSS at the last followup were 84.2%, 86.3%, 89.5%, and 83.0%, respectively. The difference between IPSS baseline and peak was greater for larger preimplant prostate volumes (≥25 mL, p = 0.004). The time to resolution was longer for higher biologic effective dose (BED) (≥210 Gy, p = 0.019 [IPSS]), in those with larger prostate volumes (≥25 mL, p = 0.025 [OABSS]), in younger patients (younger than 70 years, p = 0.043 [IPSS-S]), and in those with androgen deprivation therapy (ADT) use (p = 0.049 [IPSS-V]). Urge incontinence, included in the OABSS, was observed more commonly in older patients (75 years and older, p = 0.018), with ADT use (p < 0.001), and for higher BED (≥210 Gy, p = 0.006). CONCLUSIONS: The IPSS and OABSS showed similar patterns of change. Urinary symptoms improved more rapidly in those with high baseline IPSS levels. The OABSS was useful for following urinary symptoms after prostate brachytherapy. Age, ADT use, preimplant prostate volume, and BED were significantly associated with urinary outcomes.

Thermal unfolding process of dihydrolipoamide dehydrogenase studied by fluorescence spectroscopy.

The thermal unfolding pathway for dihydrolipoamide dehydrogenase (LipDH) isolated from Bacillus stearothermophilus was investigated focusing on the transient intermediate state characterized through time-resolved fluorescence studies. The decrease in ellipticity in the far UV region in the CD spectrum, the fluorescence spectral change of Trp-91 and FAD, and the thermal enzymatic inactivation curve consistently demonstrated that LipDH unfolded irreversibly on heat treatment at higher than 65 degrees C. LipDH took a transient intermediate state during the thermal unfolding process which could refold back into the native state. In this state, the internal rotation of FAD was activated in the polypeptide cage and correspondingly LipDH showed a peculiar conformation. The transient intermediate state of LipDH characterized in time-resolved fluorescence depolarization studies showed very similar properties to the molten-globule state, which has been confirmed in many studies on protein folding.

Roles of peptide-peptide charge interaction and lipid phase separation in helix-helix association in lipid bilayer.

The roles of peptide-peptide charged interaction and lipid phase separation in helix-helix association in lipid bilayers were investigated using a model peptide, P(24), as a transmembrane alpha-helical peptide, and its four analogues. Fluorescence amino acids, tryptophan (P(24)W) and pyrenylalanine (P(24)Pya), were introduced into the sequence of P(24), respectively. Association of these peptides permits the resonance excitation energy transfer between tryptophan in P(24)W and pyrenylalanine in P(24)Pya or excimer formation between P(24)Pya themselves. To evaluate the effect of charged interaction on the association between alpha-helical transmembrane segments in membrane proteins, charged amino acids, glutamic acid (P(24)EW) and lysine (P(24)KPya), were introduced into P(24)W and P(24)Pya, respectively. Energy transfer experiments indicated that the charged interaction between the positive charge of lysine residue in P(24)KPya and the negative charge of glutamic acid residue in P(24)EW did not affect the aggregation of transmembrane peptides in lipid membranes. As the content ratio of sphingomyelin (SM) and cholesterol (Ch) was increased in the egg phosphatidylcholine (PC), the stronger excimer fluorescence spectra of P(24)Pya were observed, indicating that the co-existence of SM and Ch in PC liposomes, that is, the raft of SM and Ch, promotes the aggregation of the alpha-helical transmembrane peptides in lipid bilayers. Since the increase in the contents of SM and Ch leads to the decrease in the content of liquid crystalline-order phase, the moving area of transmembrane peptides might be limited in the liposomes, resulting in easy formation of the excimer in the presence of the lipid-raft.

Comparison of genitourinary and gastrointestinal toxicity among four radiotherapy modalities for prostate cancer: Conventional radiotherapy, intensity-modulated radiotherapy, and permanent iodine-125 implantation with or without external beam radiotherapy.

PURPOSE: To compare late genitourinary (GU) and gastrointestinal (GI) toxicity following different prostate cancer treatment modalities. MATERIALS AND METHODS: This study included 1084 consecutive prostate cancer patients treated with conventional radiotherapy, intensity-modulated radiotherapy (IMRT), permanent iodine-125 implantation (PI) alone, and PI combined with external beam radiotherapy (PI+EBRT). The effects of treatment- and patient-related factors on late grade ⩾ 2 (G2+) GU/GI toxicity risk were assessed. RESULTS: The median follow-up was 43 months (range, 12-97 months). Compared to the PI+EBRT, there was significantly less G2+ GU toxicity in the conventional radiotherapy (hazard ratio [HR] = 0.39; 95% CI, 0.20-0.77) and the IMRT (HR=0.45, 95% CI, 0.27-0.73). Compared to the PI+EBRT, there was significantly more G2+ GI toxicity in the IMRT (HR = 2.38; 95% CI, 1.16-4.87). In PI-related groups, prostate equivalent dose in 2 Gy fractions was a significant predictor of G2+ GU toxicity (p = 0.001), and the rectal volume receiving more than 100% of the prescribed dose was a significant predictor of G2+ GI toxicity (p = 0.001). CONCLUSION: The differences in the late G2+ GU/GI risk cannot be explained by the differences in treatment modalities themselves, but by the total radiation dose to the GU/GI tract, which had a causal role in the development of late G2+ GU/GI toxicity across all treatment modality groups.

Combined brachytherapy and external beam radiotherapy without adjuvant androgen deprivation therapy for high-risk prostate cancer.

BACKGROUND: To report the outcomes of patients treated with combined iodine-125 (I-125) brachytherapy and external beam radiotherapy (EBRT) for high-risk prostate cancer. METHODS: Between 2003 and 2009, I-125 permanent prostate brachytherapy plus EBRT was performed for 206 patients with high-risk prostate cancer. High-risk patients had prostate-specific antigen ≥ 20 ng/mL, and/or Gleason score ≥ 8, and/or Stage ≥ T3. One hundred and one patients (49.0%) received neoadjuvant androgen deprivation therapy (ADT) but none were given adjuvant ADT. Biochemical failure-free survival (BFFS) was determined using the Phoenix definition. RESULTS: The 5-year actuarial BFFS rate was 84.8%. The 5-year cause-specific survival and overall survival rates were 98.7% and 97.6%, respectively. There were 8 deaths (3.9%), of which 2 were due to prostate cancer. On multivariate analysis, positive biopsy core rates and the number of high-risk factors were independent predictors of BFFS. The 5-year BFFS rates for patients in the positive biopsy core rate <50% and ≥ 50% groups were 89.3% and 78.2%, respectively (p = 0.03). The 5-year BFFS rate for patients with the any single high-risk factor was 86.1%, compared with 73.6% for those with any 2 or all 3 high-risk factors (p = 0.03). Neoadjuvant ADT did not impact the 5-year BFFS. CONCLUSIONS: At a median follow-up of 60 months, high-risk prostate cancer patients undergoing combined I-125 brachytherapy and EBRT without adjuvant ADT have a high probability of achieving 5-year BFFS.

Dosimetric effects of prone and supine positions on post-implant assessments for prostate brachytherapy.

PURPOSE: Post-implant dosimetric assessment is essential for optimal care of patients receiving prostate brachytherapy. In most institutions, post-implant computed tomography (CT) is performed in the supine position. This study aimed to assess variability in dosimetric parameters with postural changes during acquisition of post-implant CT scans. MATERIAL AND METHODS: In total, 85 consecutive patients were enrolled in this study. Fifty-three patients underwent seed implantation alone, and the remaining 32 received a combination of seed implantation and external beam radiotherapy. For post-implant analyses, CT scans were obtained in two patient positions, supine and prone. To evaluate differences in dosimetric parameters associated with postural change, the dosimetric data obtained in the supine position were defined as the standard. RESULTS: The median prostate volume was 22.4 ml in the supine and 22.5 ml in the prone position (p = 0.51). The median prostate D90 was 120.1% in the supine and 120.3% in the prone position, not significantly different. The mean prostate V100 was 97.1% in the supine and 97.0% in the prone position, again not significantly different. Median rectal V100 in supine and prone positions were 0.42 ml and 0.33 ml, respectively (p < 0.01). Rectal D2cc was also significantly decreased in the prone as compared with the supine position (median, 59.1% vs. 63.6%; p < 0.01). A larger post-implant prostate volume was associated with decreased rectal doses in the prone position. CONCLUSIONS: Though there were no significant differences among prostate D90 assessments according to postural changes, our results suggest that post-implant rectal doses decreased in the prone position.