Reinterpreting dermoscopic pigment network with reflectance confocal microscopy for identification of melanoma-specific features.

BACKGROUND: Pigment network is an important dermoscopic feature for melanocytic lesions, but alterations in grid line thickness are also observed in melanomas. OBJECTIVE: To investigate features of thick, thin and mixed pigment networks at dermoscopy and their respective features at reflectance confocal microscopy (RCM) for differential diagnosis, correlated with histology. METHODS: All melanocytic lesions with histological diagnosis, evaluated between January 2010 and May 2014, were enrolled and classified according to dermoscopy evaluation of the pigment networks: thin, thick and mixed. RESULTS: Thin network in melanoma was characterized by a honeycombed pattern (P < 0.001), dendritic cells (P < 0.001), atypical ringed pattern (P = 0.035) and structureless area (P = 0.012), whereas round cells (P < 0.001), dendritic cells (P < 0.001) and atypical meshwork pattern (<0.001) characterized thick network in melanoma. Mixed network type in melanoma shared honeycombed (P = 0.049) and typical ringed patterns (P = 0.045) in the thin area and round cells (P < 0.001) and atypical meshwork pattern (P < 0.001) in the thick area. Thin network in nevi was characterized by cobblestone (P < 0.001) and typical ringed patterns (P = 0.035), whereas thick network in nevi showed a typical meshwork pattern (P < 0.001). Mixed nevi shared the same features and patterns, but more frequently with inflammatory infiltrate (P = 0.047). CONCLUSION: Differential diagnosis between melanocytic lesions (nevi or melanoma) in thin, thick and mixed pigment networks observed at dermoscopy can be assisted by RCM to improve diagnostic accuracy.

Improving diagnostic sensitivity of combined dermoscopy and reflectance confocal microscopy imaging through double reader concordance evaluation in telemedicine settings: A retrospective study of 1000 equivocal cases.

BACKGROUND: Reflectance confocal microscopy (RCM) is an imaging device that permits non-invasive visualization of cellular morphology and has been shown to improve diagnostic accuracy of dermoscopically equivocal cutaneous lesions. The application of double reader concordance evaluation of dermoscopy-RCM image sets in retrospective settings and its potential application to telemedicine evaluation has not been tested in a large study population. OBJECTIVE: To improve diagnostic sensitivity of RCM image diagnosis using a double reader concordance evaluation approach; to reduce mismanagement of equivocal cutaneous lesions in retrospective consultation and telemedicine settings. METHODS: 1000 combined dermoscopy-RCM image sets were evaluated in blind by 10 readers with advanced training and internship in dermoscopy and RCM evaluation. We compared sensitivity and specificity of single reader evaluation versus double reader concordance evaluation as well as the effect of diagnostic confidence on lesion management in a retrospective setting. RESULTS: Single reader evaluation resulted in an overall sensitivity of 95.2% and specificity of 76.3%, with misdiagnosis of 8 melanomas, 4 basal cell carcinomas and 2 squamous cell carcinomas. Combined double reader evaluation resulted in an overall sensitivity of 98.3% and specificity of 65.5%, with misdiagnosis of 1 in-situ melanoma and 2 basal cell carcinomas. CONCLUSION: Evaluation of dermoscopy-RCM image sets of cutaneous lesions by single reader evaluation in retrospective settings is limited by sensitivity levels that may result in potential mismanagement of malignant lesions. Double reader blind concordance evaluation may improve the sensitivity of diagnosis and management safety. The use of a second check can be implemented in telemedicine settings where expert consultation and second opinions may be required.

Dermoscopy and in vivo confocal microscopy are complementary techniques for diagnosis of difficult amelanotic and light-coloured skin lesions.

BACKGROUND: Amelanotic melanomas are often difficult to diagnose. OBJECTIVES: To find and test the best methods of diagnosis using dermoscopy and reflectance confocal microscopy (RCM) tools. METHODS: We selected consecutive, difficult-to-diagnose, light-coloured and amelanotic skin lesions from three centres (in Australia and Italy). Dermoscopy and RCM diagnostic utility were evaluated under blinded conditions utilizing 45 melanomas (16 in situ, 29 invasive), 68 naevi, 48 basal cell carcinomas (BCCs), 10 actinic keratoses, 10 squamous cell carcinomas (SCCs) and 13 other benign lesions. RESULTS: Sensitivity and specificity for melanoma with dermoscopy pattern analysis by two blinded observers and their 'confidence in diagnosis' were low. The amelanotic dermoscopy method had the highest sensitivity (83%) for a diagnosis of malignancy (melanoma, BCC or SCC), but specificity was only 18%. Multivariate analysis confirmed the utility of RCM features previously identified for the diagnosis of BCC and melanoma (highest odds ratio for melanoma: epidermal disarray, dark and/or round pagetoid cells). RCM sensitivity was 67% and 73% for melanoma and BCC diagnosis, respectively, and its specificity for nonmalignant lesion diagnosis was 56%. RCM reader confidence was higher than for dermoscopy; 84% of melanomas would have been biopsied and biopsy avoided in 47% of benign lesions. All melanomas misclassified by either dermoscopy or RCM were detected by the other tool. CONCLUSIONS: Dermoscopy and RCM represent complementary/synergistic methods for diagnosis of amelanotic/light-coloured skin lesions.

Cost-benefit of reflectance confocal microscopy in the diagnostic performance of melanoma.

BACKGROUND: The sub-optimal diagnostic accuracy for melanoma leads to excise a high number of benign lesions, with consequent costs. Reflectance confocal microscopy (RCM) improves diagnostic specificity, thus possibly inducing a reduction in unnecessary excisions and related costs. OBJECTIVE: To estimate the influence of RCM on number of benign lesions needed to excise (NNE) a melanoma, in term of clinical outcomes and costs per patient. PATIENTS AND METHODS: Skin neoplasms excised by the dermatology public service in the Province of Modena were retrieved form centralized pathology database. Differences in NNE between the territorial service (using dermoscopy only) and the University Hospital (adding also RCM to the patients' workflow) were calculated and cost analysis was performed through a micro-costing approach. RESULTS: A large reduction in benign lesions excised at University Hospital was evident, leading to NNE of 6.25 for University Hospital, compared to 19.41 for Territorial Dermatology. Since 4320 unnecessary excisions can be saved every million inhabitants, an overall yearly saving of over 280,000 Eur can be expected from the use of RCM. CONCLUSIONS: The systematic use of RCM was dramatically affecting the number of benign lesions excised, and this can be translated in a significant cost-benefit advantage.

Grading keratinocyte atypia in actinic keratosis: a correlation of reflectance confocal microscopy and histopathology.

BACKGROUND: Actinic Keratosis (AK) is the clinical manifestation of cutaneous dysplasia of epidermal keratinocytes, with progressive trend towards squamous cell carcinoma. OBJECTIVE: To evaluate the strength of the correlation between keratinocyte atypia, as detected by Reflectance Confocal Microscopy (RCM) and histopathology, and to develop a more objective atypia grading scale for RCM quantification, through a discrete ranking. METHODS: A total of 48 AKs and two control areas (photodamaged and non-photodamaged skin) were selected for this study. All these areas were documented by RCM and biopsied for histopathology. One representative image of the epidermis was selected for RCM and for histopathology and used for side-by-side comparison with purpose written software. The assessor chose which of two images displayed more keratinocyte atypia, and an ordered list from the image showing the least to the most keratinocyte atypia was generated. Three evaluations were obtained for RCM and two for histopathology. RESULTS: Good interobserver correlation was obtained for RCM and histopathology grading, with high concordance between RCM and histopathology grading. CONCLUSIONS: Expert rater scan consistently distinguish different grades of cytological atypia. Non-invasive RCM data from in vivo imaging can be graded for keratinocyte atypia, comparable to histopathological grading.

Hyporeflective pagetoid cells: a new clue for amelanotic melanoma diagnosis by reflectance confocal microscopy.

BACKGROUND: Amelanotic melanoma represents a diagnostic challenge both clinically and dermoscopically. Few studies based on case series have explored the possibility of using reflectance confocal microscopy (RCM) to diagnose amelanotic melanoma. OBJECTIVES: To validate a new confocal feature, named hyporeflective pagetoid cells (HPCs), for the diagnosis of amelanotic melanoma. METHODS: A group of 20 amelanotic melanomas and a control population of nonpigmented melanocytic naevi (10), hypo/nonpigmented nonmelanocytic lesions (20) and pigmented melanomas (20), imaged by RCM, were retrospectively evaluated. The presence of HPCs and other diagnosis-specific confocal features was assessed and correlated with histopathology. RESULTS: HPCs were present, and usually abundant, in the majority of amelanotic melanomas (85%). As expected, they were also observed in Spitz naevi. On histopathology, they were correlated with pagetoid infiltration of hypomelanotic melanocytes in all melanocytic lesions. Few nonmelanocytic lesions (three squamous cell carcinomas, two seborrhoeic keratoses and one basal cell carcinoma) showed the presence of HPCs. In these cases, they corresponded to enlarged or dyskeratotic keratinocytes by histopathology. CONCLUSIONS: The identification of HPCs in the epidermis is a new parameter that is frequently found in amelanotic melanoma. Possible confounders are represented by atypical keratinocytes that can be present in nonmelanocytic lesions. However, the whole architecture and the presence of additional diagnostic criteria should be considered in order to obtain a correct diagnosis.

Fibrosis-related gene expression in the prostate is modulated by doxazosin treatment.

AIMS: To gain new insights into the molecular mechanisms of action of doxazosin, we investigated the prostatic stroma ultrastructure and the expression of genes involved with fibrosis, such as collagen type I and III (COL1A1 and COL3A1, respectively) and TGF-beta 1, in the rat ventral prostate. MAIN METHODS: Adult Wistar rats were treated with doxazosin (25mg/kg/day), and the ventral prostates were excised at 7 and 30days after treatment. Untreated rats were controls. Ventral prostates were subjected to ultrastructural, immunohistochemical, biochemical and molecular analyses. KEY FINDINGS: Doxazosin-treated prostates showed thickened bundles of collagen fibrils, activated fibroblasts, enlarged neurotransmitter vesicles and increased tissue immunostaining for collagen type I and type III when compared to untreated prostates. After 7 and 30days of doxazosin treatment mRNA expression of COL1A1 and COL3A1 was significantly increased and reduced, respectively, compared to the control group. TGF-beta 1 mRNA and protein levels were increased after 7days of doxazosin treatment, whereas only mRNA levels remained increased after 30days of treatment. SIGNIFICANCE: Our data suggest that relaxation of smooth muscle cells by alpha-blockers interferes with the mechanical dynamics of the prostatic stroma extracellular matrix components, generating a pro-fibrotic effect probably via the TGF-beta 1 signaling pathway. Long term treatment with doxazosin may also lead to a reduced turnover of extracellular matrix components. Our results add to a better understanding of the molecular mechanisms behind the effects of alpha-blockade on prostatic histoarchitecture and the response to treatment for benign prostatic hyperplasia.

Novel blue light-sensitive proteins from a metagenomic approach.

A microarray-based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor-encoding genes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2-5 microg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 microg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid-cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per-Arnt-Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht-met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht-met1 is followed by a second open reading frame encoding a Fe-chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht-met1 was subcloned and expressed yielding a fully functional, flavin-containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin-protein photoadduct. The dark recovery lifetime was found as tau(REC) = 120 s (20 degrees C) and is among the fastest ones determined so far for bacterial LOV domains.

99Tc nanocolloid sentinel node procedure in papillary thyroid carcinoma: our mono-institutional experience on a large series of patients.

The sentinel lymph node was defined as the first lymph node to receive drainage from a primary cancer. The aims of this study were to investigate the efficacy of radiocolloid lymphoscintigraphy and of the hand-held gamma probe procedure for sentinel lymph node biopsy in papillary thyroid carcinoma and to evaluate these results in clinical staging. A total of 99 consecutive papillary thyroid carcinoma patients entered the study. Patients underwent radiocolloid lymphoscintigraphy before surgery. Intra-operative sentinel lymph node localization was performed using a hand-held gamma probe. Patients were observed at follow-up at 2 and 6 months and, thereafter, yearly. Sequential lymphoscintigraphy was able to identify at least one sentinel lymph node in 98/99 cases (99%), using intra-operative hand-held gamma probe, the surgeon was able to detect at least one sentinel lymph node in all cases. Sentinel lymph node metastases were diagnosed in 49%. Overall, 79 patients underwent ablative (131)I therapy. The median value of thyroglobulin in N0 vs. N1 patients was 1 ng/ml vs. 1.9 ng/ml (p = 0.03) and 0.2 ng/ml vs. 1 ng/ml (p = 0.001) before and after (131)I therapy, respectively. The pre-operative lymphoscintigraphy and the intra-operative gamma probe offer significant advantages over the vital dye technique, described in our previous experience. The rate of nodal involvement (49%) is very high considering that no patients had clinically palpable nodes or suspected at echography. (131)I whole body scan and thyroglobulin measurements confirmed sentinel lymph node in papillary thyroid carcinoma as a reliable procedure. In patients classified N0, by sentinel lymph node biopsy, ablative (131)I therapy could be avoided.

Bacterial bilin- and flavin-binding photoreceptors.

The growing number of sequenced prokaryotic genomes reveals a wide distribution of open reading frames (ORFs) that putatively encode for red- and blue light sensing photoreceptors. They comprise the bilin-binding phytochromes and the flavin-binding cryptochromes, LOV and BLUF proteins, indicating that about 1/4 of bacteria do possess at least one of these photosensory proteins. The distribution of red- and blue-light sensors among different prokaryotic phyla and classes, and their functional activity as light-switched systems are the subject of this perspective. These photoreceptors were originally found in plants by following the associated physiological responses induced by the respective spectral irradiation. Genome-based approaches now require the assignment of a photochemical/physiological function to the heterologously expressed gene product. Database searches demonstrate in some cases several genes of one category in a certain prokaryot, indicating the presence of more than one type of red- or blue-light sensing properties, but also show a combination of proteins with both spectral sensitivities. Another interesting feature now "comes into light": according to their nature as biological sensors, these photoreceptors are equipped with signalling domains, initiating a cellular response, thereby constituting modular systems switchable by light. It is seen that many of these signalling domains, now found together with light-inducible sensing domains, were already described for other stimuli, e.g., osmo-regulation, oxygen, hydrogen, chemicals, or pH. In some cases, the same type of signalling domain can be found in a red- or a blue-light sensing photoreceptor. Following the characterization of their photochemistry, for several of these bacterial photoreceptors physiological functions are now assigned.

A blue light inducible two-component signal transduction system in the plant pathogen Pseudomonas syringae pv. tomato.

The open reading frame PSPTO2896 from the plant pathogen Pseudomonas syringae pv. tomato encodes a protein of 534 amino acids showing all salient features of a blue light-driven two-component system. The N-terminal LOV (light, oxygen, voltage) domain, potentially binding a flavin chromophore, is followed by a histidine kinase (HK) motif and a response regulator (RR). The full-length protein (PST-LOV) and, separately, the RR and the LOV+HK part (PST-LOV(DeltaRR)) were heterologously expressed and functionally characterized. The two LOV proteins showed typical LOV-like spectra and photochemical reactions, with the blue light-driven, reversible formation of a covalent flavin-cysteine bond. The fluorescence changes in the lit state of full-length PST-LOV, but not in PST-LOV(DeltaRR), indicating a direct interaction between the LOV core and the RR module. Experiments performed with radioactive ATP uncover the light-driven kinase activity. For both PST-LOV and PST-LOV(DeltaRR), much more radioactivity is incorporated when the protein is in the lit state. Furthermore, addition of the RR domain to the fully phosphorylated PST-LOV(DeltaRR) leads to a very fast transfer of radioactivity, indicating a highly efficient HK activity and a tight interaction between PST-LOV(DeltaRR) and RR, possibly facilitated by the LOV core itself.

Initial characterization of a blue-light sensing, phototropin-related protein from Pseudomonas putida: a paradigm for an extended LOV construct.

The open reading frame PP2739 from Pseudomonas putida KT2440 encodes a 151 amino acid protein with sequence similarity to the LOV domains of the blue-light sensitive protein YtvA from Bacillus subtilis and to the phototropins (phot) from plants. This sensory box LOV protein, PpSB2-LOV, comprises a LOV core, followed by a C-terminal segment predicted to form an alpha-helix, thus constituting a naturally occurring paradigm for an extended LOV construct. The recombinant PpSB2-LOV shows a photochemistry very similar to that of YtvA and phot-LOV domains, yet the lifetime for the recovery dark reaction, taurec=114 s at 20 degrees C, resembles that of phot-LOV domains (5-300 s) and is much faster than that of YtvA or YtvA-LOV (>3000 s). Time-resolved optoacoustics reveals phot-like, light-driven reactions on the ns-micros time window with the sub-nanosecond formation of a flavin triplet state (PhiT=0.46) that decays into the flavin-cysteine photoadduct with 2 micros lifetime (Phi390=0.42). The fluorescence spectrum and lifetime of the conserved W97 resembles the corresponding W103 in full-length YtvA, although the quantum yield, PhiF, is smaller (about 55% of YtvA) due to an enhanced static quenching efficiency. The anisotropy of W97 is the same as for W103 in YtvA (0.1), and considerably larger than the value of 0.06, found for W103 in YtvA-LOV. Different to YtvA and YtvA-LOV, the fluorescence for W97 becomes larger upon photoproduct formation. These data indicate that W97 is located in a similar environment as W103 in full-length YtvA, but undergoes larger light-driven changes. It is concluded that the protein segment located C-terminally to the LOV core (analogous to an interdomain linker) is enough to confer to the conserved tryptophan the fluorescence characteristics typical of full-length YtvA. The larger changes experienced by W97 upon light activation may reflect a larger conformational freedom of this protein segment in the absence of a second domain.

Non-HDL cholesterol predicts coronary heart disease in primary prevention: findings from an Italian 40-69 year-old cohort in general practice.

OBJECTS: Non-HDL cholesterol is now recommended as an index of risk associated with combined dyslipidemia, and it has also been found useful in predicting coronary heart disease (CHD) risk in patients with diabetes. We studied the association between known CHD risk factors, enclosed non-HDL cholesterol, and a "high CHD risk condition", i.e. a "5-years CHD risk >15%" in general practice. METHODS: We studied 4,085 40-69 year-old diabetic (no. 489) and non-diabetic (no. 3,596) individuals from an opportunistic cohort. Cross-sectional descriptive statistics, and age- and gender-adjusted multiple logistic exponential betas have been calculated. RESULTS: About 12% of the participants had diabetes. Age- and gender-adjusted comparison showed that all the study variables were significantly worse in diabetic vs. non-diabetic individuals (except cigarette smoking, total blood cholesterol and the ratio of total to HDL cholesterol). They had a mean "5-year CHD-risk" significantly higher than non-diabetic individuals (18.8+/-11.9% vs 7.5+/-6.9%, P<0.01), and a four-fold prevalence of "5-years CHD risk >15%" (55.4% vs 11.1%, P<0.01). As to diabetic individuals, the study variables associated to a "high CHD risk condition" were cigarette smoking, systolic blood pressure, and non-HDL blood cholesterol levels. As to non-diabetic individuals cigarette smoking, systolic blood pressure, and HDL (inversely) and non-HDL blood cholesterol levels were associated to a "high CHD risk condition". CONCLUSIONS: Non-HDL cholesterol--and cigarette smoking and systolic blood pressure--strongly predicted a "high CHD risk condition" both in diabetic and non-diabetic individuals.

Functional variations among LOV domains as revealed by FT-IR difference spectroscopy.

The two LOV domains, LOV1 and LOV2, from Chlamydomonas reinhardtii were investigated by light-induced FT-IR difference spectroscopy and compared to the LOV domain of Bacillus subtilis (YtvA-LOV). It is shown that the two S-H conformations of the reactive LOV1 cysteine C57(1) are exposed to environments of different hydrogen bonding strength. Thus, the two rotamer configurations of C57 might be related to the fact that the triplet state decays bi-exponentially into the LOV1-390 photoproduct. Exchange of the two other cysteines of LOV1 (C32S and C83S) does not alter the S-H stretching band providing evidence that this band feature arises solely from C57. The reactive cysteine of LOV2 from Chlamydomonas reinhardtii (C250) and of YtvA-LOV (C62) exhibit both a homogenous S-H stretching vibrational band which suggests a single conformer of the amino acid side chain. Finally, the FT-IR difference spectrum of YtvA from Bacillus subtilis comprising the light absorbing LOV domain and the putative signaling STAS (sulfate transporter/antisigma-factor antagonist) domain, reveals conformational changes in the latter after blue-light excitation.

Thermodynamics of the early steps in the photocycle of Natronobacterium pharaonis halorhodopsin. Influence of medium and of anion substitution.

The enthalpy (delta H) and structural volume changes (delta V) associated with the formation and decay of the early intermediate K600 in the photocycle of Natronobacterium pharaonis halorhodopsin (pHR), an inward-directed anion pump, were obtained by laser-induced optoacoustic spectroscopy. A large expansion is associated with K600 formation, its value depending on the medium and on the anion (Cl-, NO3-, Br-, I-). A smaller expansion is associated with K600 decay to L520. A contraction is found for the same step in the case of the azide-loaded pHR which is an efficient outward-directed proton pump. Thus, the conformational changes in L520 determine the direction and sign of charge translocation. The linear correlation between delta H and delta V for chloride-loaded pHR observed upon mild medium variations is attributed to enthalpy-entropy compensation effects and allows the calculation of the free-energy changes, delta GK = (97 +/- 16) kJ/mol and delta GKL = -(2 +/- 2) kJ/mol. Different from other systems, delta S correlates negatively with delta V in the first steps of the pHR photocycle. Thus, the space around the anion becomes larger and more rigid during each of these two steps. The photocycle quantum yield was 0.52 for chloride-pHR as measured by laser flash photolysis.

Time-resolved thermodynamic changes photoinduced in 5,12-trans-locked bacteriorhodopsin. Evidence that retinal isomerization is required for protein activation.

Structural volume changes upon excitation of isomerization-blocked 5,12-trans-locked bacteriorhodopsin (bR) (bacterio-opsin + 5-12-trans-locked retinal) were studied using photothermal methods. The very small prompt expansion detected using laser-induced optoacoustics (0.3 mL/mol of absorbed photons) is assigned to a charge reorganization in the chromophore protein pocket concomitant with the formation of the intermediate T5.12. The subsequent contraction associated with a 300 ns lifetime is assigned to protein movements required to reach the entire chromoprotein free energy minimum, after the 17 ps optical decay of T5.12. The volume changes comprise the entropy of medium rearrangement during T5.12 formation and decay. The slow changes detected in previous studies by atomic force microscopy might be explained by the slowing down of movements in films containing 5,12-trans-locked bR. Photothermal beam deflection data with the 5,12-trans-locked bR suspensions indicate no further changes in microseconds to hundreds of milliseconds. Thus, all the absorbed energy is either released to the solution as heat or used for entropy changes within the first 300 ns after the pulse, supporting the paradigm that isomerization is required for signal transduction in retinal proteins. Bacterio-opsin assembled with all-trans-retinal afforded (similar to data reported with wild-type bR) an expansion of 2.6 mL/mol (assigned to the production of KE) followed by a further expansion of 0.8 mL/mol (KE-->KL; KE, KL, early and late K's) involving no heat loss. For KL decay to L, a contraction of 6 mL/mol of phototransformed reconstituted all-trans bR was determined.

Aspartate 75 mutation in sensory rhodopsin II from Natronobacterium pharaonis does not influence the production of the K-like intermediate, but strongly affects its relaxation pathway.

The early steps in the photocycle of the aspartate 75-mutated sensory rhodopsin II from Natrobacterium pharaonis (pSRII-D75N) were studied by time-resolved laser-induced optoacoustic spectroscopy combined with quantum yield determinations by flash photolysis with optical detection. Similar to the case of pSRII-WT, excitation of pSRII-D75N produces in subnanosecond time a K-like intermediate. Different to the case of K in pSRII-WT, in pSRII-D75N there are two K states. K(E) decays into K(L) with a lifetime of 400 ns (independent of temperature in the range 6.5-52 degrees C) which is optically silent under the experimental conditions of our transient absorption experiments. This decay is concomitant with an expansion of 6.5 ml/mol of produced intermediate. This indicates a protein relaxation not affecting the chromophore absorption. For pSRII-D75N reconstituted into polar lipids from purple membrane, the mutation of Asp-75 by the neutral residue Asn affects neither the K(E) production yield (PhiK(e) 0.51 +/- 0.05) nor the energy stored by this intermediate (E(E)K(E) = 91 +/- 11 kJ/mol), nor the expansion upon its production (DeltaV(R,1) = 10 +/- 0.3 ml/mol). All these values are very similar to those previously determined for K with pSRII-WT in the same medium. The millisecond transient species is attributed to K(L) with a lifetime corresponding to that determined by electronic absorption spectroscopy for K(565). The determined energy content of the intermediates as well as the structural volume changes for the various steps afford the calculation of the free energy profile of the phototransformation during the pSRII-D75N photocycle. These data offer insights regarding the photocycle in pSRII-WT. Detergent solubilization of pSRII-D75N affects the sample properties to a larger extent than in the case of pSRII-WT.