Spatiotemporal analysis of lake chlorophyll-a with combined in situ and satellite data.

We estimated chlorophyll-a (Chl-a) concentration using various combinations of routine sampling, automatic station measurements, and MERIS satellite images. Our study site was the northern part of the large, shallow, mesotrophic Lake Pyhäjärvi located in southwestern Finland. Various combinations of measurements were interpolated spatiotemporally using a data fusion system (DFS) based on an ensemble Kalman filter and smoother algorithms. The estimated concentrations together with corresponding 68% confidence intervals are presented as time series at routine sampling and automated stations, as maps and as mean values over the EU Water Framework Directive monitoring period, to evaluate the efficiency of various monitoring methods. The mean Chl-a calculated with DFS in June-September was 6.5-7.5 µg/l, depending on the observations used as input. At the routine monitoring station where grab samples were used, the average uncertainty (standard deviation, SD) decreased from 2.7 to 1.6 µg/l when EO data were also included in the estimation. At the automatic station, located 0.9 km from the routine monitoring site, the SD was 0.7 µg/l. The SD of spatial mean concentration decreased from 6.7 to 2.9 µg/l when satellite observations were included in June-September, in addition to in situ monitoring data. This demonstrates the high value of the information derived from satellite observations. The conclusion is that the confidence of Chl-a monitoring could be increased by deploying spatially extensive measurements in the form of satellite imaging or transects conducted with flow-through sensors installed on a boat and spatiotemporal interpolation of the multisource data.

Endotoxemia, nutrition, and cardiometabolic disorders.

AIMS: Circulating lipopolysaccharides (LPSs), associated with both infection and inflammation, may arise from the gastrointestinal tract microbiota, and the levels may be affected by daily nutrition. We investigated whether nutrient intake affects the association of serum LPS activity with prevalent obesity, metabolic syndrome (MetS), diabetes, and coronary heart disease (CHD) and with the risk of incident CHD events. METHODS: The nutrition cohort (n = 2,452, mean age ± SD, 52.2 ± 10.1 years) of the FINRISK 1997 Study was followed up for 10 years. Information on macronutrient intake at baseline was collected from 24-h dietary recall. Serum endotoxin activities were determined by the Limulus amebocyte lysate assay. RESULTS: LPS activity was associated directly with the total energy intake and indirectly with carbohydrate intake in lean, healthy subjects. High LPS was significantly associated with prevalent obesity, MetS, diabetes, and CHD events, independently of established risk factors, CRP, and total energy or nutrient intake. The ORs (95 % CI) were 1.49 (1.21-1.85, p < 0.001, Q2-4 vs. Q1) for obesity, 2.56 (1.97-3.32, p < 0.001, Q2-4 vs. Q1) for MetS, 1.94 (1.06-3.52, p = 0.031, Q2-4 vs. Q1) for CHD, and 1.01 (1.00-1.01, p = 0.032, LPS unit) for diabetes. In the follow-up, high LPS was significantly associated with the risk of CHD events with a hazard ratio of 1.88 (1.13-3.12, p = 0.013, Q2-4 vs. Q1). This association was independent of baseline established risk factors, diet, obesity, MetS, and diabetes. CONCLUSION: A high serum LPS activity is strongly associated with cardiometabolic disorders, which supports the role of bacterial infections and immune response in their etiology.

Genetic variation on the BAT1-NFKBIL1-LTA region of major histocompatibility complex class III associates with periodontitis.

Periodontitis is a chronic inflammatory disease with a multifactorial etiology. We investigated whether human major histocompatibility complex (MHC) polymorphisms (6p21.3) are associated with periodontal parameters. Parogene 1 population samples (n = 169) were analyzed with 13,245 single nucleotide polymorphisms (SNPs) of the MHC region. Eighteen selected SNPs (P ≤ 0.001) were replicated in Parogene 2 population samples (n = 339) and the Health 2000 Survey (n = 1,420). All subjects had a detailed clinical and radiographic oral health examination. Serum lymphotoxin-α (LTA) concentrations were measured in the Parogene populations, and the protein was detected in inflamed periodontal tissue. In the Parogene 1 population, 10 SNPs were associated with periodontal parameters. The strongest associations emerged from the parameters bleeding on probing (BOP) and a probing pocket depth (PPD) of ≥6 mm with the genes BAT1, NFKBIL1, and LTA. Six SNPs, rs11796, rs3130059, rs2239527, rs2071591, rs909253, and rs1041981 (r(2), ≥0.92), constituted a risk haplotype. In the Parogene 1 population, the haplotype had the strongest association with the parameter BOP, a PPD of ≥6 mm, and severe periodontitis with odds ratios (95% confidence intervals) of 2.63 (2.21 to 3.20), 2.90 (2.37 to 3.52), and 3.10 (1.63 to 5.98), respectively. These results were replicated in the other two populations. High serum LTA concentrations in the Parogene population were associated with the periodontitis risk alleles of the LTA SNPs (rs909253 and rs1041981) of the haplotype. In addition, the protein was expressed in inflamed gingival connective tissue. We identified a novel BAT1-NFKBIL1-LTA haplotype as a significant contributor to the risk of periodontitis. The genetic polymorphisms in the MHC class III region may be functionally important in periodontitis susceptibility.

Very low density lipoproteins derived from periodontitis patients facilitate macrophage activation via lipopolysaccharide function.

OBJECTIVE: Periodontitis, a chronic oral infection caused mainly by gram-negative bacteria, induces endotoxemia and associates with the risk for atherosclerosis. We investigated the effect of periodontal treatment on proatherogenic properties of very low density lipoproteins (VLDL). METHODS: VLDL were isolated from 30 systemically healthy periodontitis patients before (pre-treatment) and 3 months after treatment (post-treatment). The mass compositions were analyzed, and VLDL-induced changes in cellular cholesterol content and expression of selected genes of human THP-1 macrophages were measured. RESULTS: Periodontal treatment decreased the local inflammation in the periodontium, but did not have a significant effect on C-reactive protein (CRP) levels, VLDL composition, or VLDL potential to induce cholesterol uptake or gene expression by the macrophages. Incubation of macrophages in the presence of VLDL resulted in more than twofold increase in their cellular cholesterol content. Uptake of VLDL with ensuing macrophage cholesterol accumulation correlated positively with VLDL-associated lipopolysaccharide (LPS) activity (r=0.436, P=.016) and apolipoprotein E content (r=0.374, P=.046). Pre-treatment VLDL derived from the patients with high CRP levels displayed higher LPS activity than that of VLDL derived from patients with low CRP (above vs. below median, P=.007). In addition, pre-treatment VLDL isolated from patients with high systemic inflammation induced higher relative mRNA expression of CD14, TNF-α, MCP-1, and IL-6 in the macrophages. CONCLUSION: Inflammation and endotoxemia induced by severe periodontitis may increase VLDL-dependent macrophage activation and cellular cholesterol accumulation, and thereby atherogenesis.

Lipopolysaccharide associates with pro-atherogenic lipoproteins in periodontitis patients.

INTRODUCTION: Periodontitis patients are known to suffer from endotoxemia, which may be among the major risk factors for atherosclerosis. In health, lipopolysaccharide (LPS) is mainly carried with high density lipoprotein (HDL) particles. Shift of LPS toward lipoproteins with lower densities may result in less effective endotoxin scavenging. Our aim was to determine plasma LPS activity and lipoprotein-distribution before and after treatment in periodontitis patients. PATIENTS AND METHODS: Very low and intermediate density (VLDL-IDL), low density (LDL), HDL 2, HDL3, and lipoprotein-deficient plasma (LPDP) were isolated by sequential ultracentrifugation. Patients included 34 subjects aged 53.5 +/- 8.3 years, before and 6 months after periodontal treatment. RESULTS: The mean LPS distribution decreased among lipoprotein classes as follows: VLDL-IDL 41.3 +/- 12.1%, LPDP 25.0 +/- 7.0%, HDL3 13.1 +/- 5.2%, LDL 11.5 +/- 3.7%, and HDL2 9.2 +/- 2.8%. Plasma and VLDL-IDL-associated LPS correlated positively, and LDL- and HDL-associated LPS negatively with clinical periodontal parameters and plasma cytokine concentrations. Mean plasma LPS activity increased after periodontal treatment from 44.0 +/- 17.0 to 55.7 +/- 24.2 EU/ml (P = 0.006). No significant changes were found in LPS lipoprotein distribution and lipoprotein compositions after the treatment. CONCLUSIONS: Endotoxemia increases with severity of periodontitis. In periodontitis, LPS associates preferentially with the pro-atherogenic VLDL-IDL fraction. Periodontal treatment has only minor effects on plasma LPS activity or distribution, which reflects persistence of the disease.

SOAP-based services provided by the European Bioinformatics Institute.

SOAP (Simple Object Access Protocol) (http://www.w3.org/TR/soap) based Web Services technology (http://www.w3.org/ws) has gained much attention as an open standard enabling interoperability among applications across heterogeneous architectures and different networks. The European Bioinformatics Institute (EBI) is using this technology to provide robust data retrieval and data analysis mechanisms to the scientific community and to enhance utilization of the biological resources it already provides [N. Harte, V. Silventoinen, E. Quevillon, S. Robinson, K. Kallio, X. Fustero, P. Patel, P. Jokinen and R. Lopez (2004) Nucleic Acids Res., 32, 3-9]. These services are available free to all users from http://www.ebi.ac.uk/Tools/webservices.

Detection of water quality using simulated satellite data and semi-empirical algorithms in Finland.

The aim of the study was to test the feasibility of the band combination of the TERRA MODIS and ENVISAT MERIS instruments for operational monitoring of lakes and coastal waters in Finland. Also simulated LANDSAT TM data were tested. Satellite bands were simulated using airborne measurements with AISA imaging spectrometer. Semi-empirical algorithms with simulated satellite data were tested against field observations using regression analysis. Interpretation of chlorophyll a, suspended matter, turbidity and secchi-disk depth was included in the analyses. The data for this study were gathered in campaigns carried out in May and August 1997 and August 1998 both for lakes in southern Finland and coastal waters of the Baltic Sea. The data set included 85 in situ observations for lakes and 107 for coastal waters. Our results show that the band combination to be included in the ENVISAT MERIS instrument enables the interpretation of water quality, including chlorophyll a concentration using semi-empirical algorithms both for lakes and coastal waters. MERIS band 9 centred at 705 nm is proven to be of vital importance for the detection of chlorophyll a in local surface waters.

A hyperspectral model for interpretation of passive optical remote sensing data from turbid lakes.

A hyperspectral model was developed for the interpretation of remote sensing data collected above inland waters. Specific absorption and scattering coefficients proposed by other authors were not suitable for modelling of the irradiance reflectance in 12 studied lakes. Therefore, special studies were carried out to estimate absorption and scattering coefficients as well as backscattering probability of suspended matter in turbid waters. AC-9 and Li-1800UW results were used for these purposes. The algorithms obtained were used to improve the model, which was then tested in forward and inverse modes.

Retrieval of water quality from airborne imaging spectrometry of various lake types in different seasons.

The suitability of the AISA airborne imaging spectrometer for monitoring lake water quality was tested in four surveys carried out in southern Finland in 1996-1998. Altogether, 11 lakes were surveyed and the total number of stations with concurrent remote sensing and limnological measurements was 127. The ranges of the water quality variables were: the sum of chlorophyll a and phaeophytin a 1-100 microg l(-1), turbidity 0.4-26 FNU, total suspended solids 0.7-32 mg l(-1), absorption coefficient of aquatic humus at 400 nm 1.2-14 m(-1) and secchi disc transparency 0.4-7 m. For the retrieval analyses, 24 AISA channels in the 450-786 nm range with a channel width of 6-14 nm were used. The agreement between estimated and observed water quality variables was generally good and R2 for the best algorithms was in the range of 0.72-0.90 over the whole dataset. The channels used for May were, in most cases, the same as those for August, but the empirical parameters of the algorithms were different. After seasonal grouping, R2 varied from 0.84 to 0.95. The use of apparent reflectance instead of radiance improved the estimation of water quality in the case of total suspended solids and turbidity. In the most humic lake, the empirical algorithms tested were suitable only for the interpretation of total suspended solids and turbidity.

A semi-operative approach to lake water quality retrieval from remote sensing data.

A semi-operative approach to retrieve chlorophyll-a concentration from airborne/spaceborne spectrometer observations has been developed and tested using the airborne imaging spectrometer (AISA) data from 11 lakes located in southern Finland. The retrieval approach is empirical and requires nearly simultaneous in situ training data on water quality for the determination of regression coefficients. However, the training data does not have to be collected from every lake under investigation. Instead, the results obtained indicate that reliable estimates on the level of chlorophyll-a (chl-a) for an individual lake can be achieved without employing in situ data representing this specific lake. This enables the estimation of water quality from remotely sensed data for numerous lakes with the aid of reference data only for a few selected lakes representing the region under investigation. In addition, it is shown that the remotely sensed spectrum shape characteristics are highly affected by the trophic and humic state of the lake water.

Analysis on the feasibility of multi-source remote sensing observations for chl-a monitoring in Finnish lakes.

Chlorophyll-a (chl-a) concentration of lake water can be measured with airborne (or spaceborne) optical remote sensing instruments. The rmse obtained here with empirical algorithms and 122 measurement points was 8.9 microg/l (all points used for training and testing). Airborne Imaging Spectrometer for Applications (AISA) was used in four lake water quality measurement campaigns (8 measurement days) in southern Finland during 1996-1998 with other airborne instruments and extensive in situ data collection. As empirical algorithms are employed for chl-a retrieval from remote sensing data, temporally varying factors such as surface reflection and atmospheric effects degrade the estimation accuracy. This paper analyzes the quantitative accuracy of empirical chl-a retrieval algorithms available as methods to correct temporal disturbances are either included or excluded. The aim is to evaluate the usability of empirical chl-a retrieval algorithms in cases when no concurrent reference in situ data are available. Four methods to reduce the effects of temporal variations are investigated. The methods are: (1) atmospheric correction; (2) synchronous radiometer data; (3) wind speed data; and (4) bidirectional scattering model based on wind speed and sun angle data. The effects of different correction methods are analyzed by using single-date test data and multi-date training data sets. The results show that the use of a bidirectional scattering model and atmospheric correction reduces the bias component of the measurement error. Radiometer data also appear to improve the accuracy. However, if concurrent in situ reference data are not available, the retrieval algorithms and correction methods should be improved for reducing the bias error.

Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution.

The crystal structure of DsRed, a red fluorescent protein from a corallimorpharian, has been determined at 2.0-A resolution by multiple-wavelength anomalous dispersion and crystallographic refinement. Crystals of the selenomethionine-substituted protein have space group P2(1) and contain a tetramer with 222 noncrystallographic symmetry in the asymmetric unit. The refined model has satisfactory stereochemistry and a final crystallographic R factor of 0.162. The protein, which forms an obligatory tetramer in solution and in the crystal, is a squat rectangular prism comprising four protomers whose fold is extremely similar to that of the Aequorea victoria green fluorescent protein despite low ( approximately 23%) amino acid sequence homology. The monomer consists of an 11-stranded beta barrel with a coaxial helix. The chromophores, formed from the primary sequence -Gln-Tyr-Gly- (residues 66-68), are arranged in a approximately 27 x 34-A rectangular array in two approximately antiparallel pairs. The geometry at the alpha carbon of Gln-66 (refined without stereochemical restraints) is consistent with an sp(2) hybridized center, in accord with the proposal that red fluorescence is because of an additional oxidation step that forms an acylimine extension to the chromophore [Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K. & Tsien, R. Y. (2000) Proc. Natl. Acad. Sci. USA 87, 11990-11995]. The carbonyl oxygen of Phe-65 is almost 90 degrees out of the plane of the chromophore, consistent with theoretical calculations suggesting that this is the minimum energy conformation of this moiety despite the conjugation of this group with the rest of the chromophore.

Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein.

The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells. Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values. At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5). To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution. The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding. A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface. Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces. Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed.

Sodium intake of 1 to 5-year-old children: the STRIP project. The Special Turku Coronary Risk Factor Intervention Project.

The aim of this study is to examine sodium intake and dietary sodium sources of 1-5-y-old children in a prospective, randomized long-term coronary heart disease prevention trial, focused on dietary fat modification. Counselling included no advice about reducing salt in the children's diets. Food consumption of 100 intervention children and 100 control children was recorded for 3 consecutive days at the age of 13 mo and for 4 consecutive days at the ages of 3 and 5 y. Sodium intakes were calculated using the Micro Nutrica program. Children's mean daily sodium (NaCl) consumption (intervention and control children combined) was 1600+/-527 mg (4.0+/-1.3 g), 1900+/-504 mg (4.8+/-1.3 g) and 2200+/-531 mg (5.5+/-1.3 g) at the ages of 13 mo and 3 and 5 y, respectively. The intervention children consumed as much or slightly more sodium than the control children at all ages studied. Half the sodium consumption was derived from added salt in commercially prepared or homemade foods. Milk, meat products, bread and cereals were other important sodium sources. In conclusion, nutrition counselling in the Special Turku Coronary Risk Factor Intervention Project (STRIP) trial, with its main focus on the quality of fat in child nutrition, has had minimal influence on children's sodium intake. To avoid excessive sodium intake in children, dietary counselling should include information about salt use, and food manufacturers should be encouraged to provide more low-sodium products.

Structural and spectral response of green fluorescent protein variants to changes in pH.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracellular pH. To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pattern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (S65G/V68L/S72A/T203Y) to change the solvent accessibility (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore. pH titrations of these variants indicate that the chromophore pKa can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9. Finally, mutagenesis was used to raise the pKa from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbestic point. This raises the interesting possibility of using fluorescence at this isosbestic point as an internal reference. Practical real time in vivo applications in cell and developmental biology are proposed.

Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein.

BACKGROUND: Because of its ability to spontaneously generate its own fluorophore, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria is used extensively as a fluorescent marker in molecular and cell biology. The yellow fluorescent proteins (YFPs) have the longest wavelength emissions of all GFP variants examined to date. This shift in the spectrum is the result of a T203Y substitution (single-letter amino acid code), a mutation rationally designed on the basis of the X-ray structure of GFP S65T. RESULTS: We have determined the crystal structures of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 A resolution, respectively. Both structures show clear electron density for nearly coplanar pi-pi stacking between Tyr203 and the chromophore. The chromophore has been displaced by nearly 1 A in comparison to other available structures. Although the H148G mutation results in the generation of a solvent channel to the chromophore cavity, intense fluorescence is maintained. The chromophore in the intact protein can be titrated, and the two variants have pKa values of 7.0 (YFP) and 8.0 (YFP H148G). CONCLUSIONS: The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizability of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial for the chemistry of chromophore formation. The YFPs significantly extend the pH range over which GFPs may be employed as pH indicators in live cells.

[Salt intake in young children]. / Saltintag hos små barn.

Among adult Finns salt intake is about twice as high as the recommended levels and almost five-fold greater than the physiological requirement. Information as to salt intake in children has hitherto been sparse. Daily sodium intake among 1-5-year-olds was investigated in this study, and the foodstuffs from which it was derived were identified. In all age groups, sodium intake was at least two-fold greater than the Nordic recommendations, and among five-year-olds it exceeded the recommended intake for adults. Approximately half of the sodium intake was found to have derived from salt used in cooking. Levels of sodium derived by children from dairy, meat and grain products were also relatively high. Sodium intake in children after infancy merits greater attention than previously accorded it, as permanent eating habits and taste preferences are already formed at this age, and reasonable limits for salt intake should therefore be established during childhood.

A new versatile PC-based lung sound analyzer with automatic crackle analysis (HeLSA); repeatability of spectral parameters and sound amplitude in healthy subjects.

A versatile PC-based lung sound analyzer has been developed for short-term recording and analysis of respiratory sounds in research and clinical applications. The system consists of two sound sensors, a flow sensor, a filtering signal amplifier and a PC with a data acquisition card and software for measurement and analysis of the sounds. The analyses include phonopneumography, time expanded waveform analysis, spectral analysis with time averaged Fast Fourier Transform, frequency analysis in time domain (sonogram), and automatic detection and waveform analysis of crackles. Short-term repeatability of spectral parameters of tracheal and lung sounds was studied in 10 healthy subjects. The coefficients of variation (CoV) of the averaged quartile frequencies (F25, F50 and F75) of lung sounds during flow-controlled tidal breathing were 3.7, 4.0 and 8.9% in expiration and 2.7, 3.5 and 4.5% in inspiration, respectively. CoVs of the averaged F25, F50 and F75 of expiratory tracheal sounds were 6.9, 3.0 and 2.4%, and those of inspiratory tracheal sounds 6.3, 2.6 and 3.3%, respectively. Examples of lung sound analysis of samples containing adventitious sounds such as crackles and wheezes are presented. The results indicate that the median frequency has the best repeatability of quartile frequencies of breath sounds and they suggest that the variations of those parameters are low enough for diagnostic purposes. The results also suggest that the analyzer can be a useful new tool for pulmonary research in the fields of physiological and clinical short-term studies of respiratory sounds.

Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein.

The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.