NONLINEAR POLARIZATION SPECTROSCOPY (FREQUENCY DOMAIN) STUDIES OF EXCITED STATE PROCESSES: THE B800–850ANTENNA OF RHODOBACTER SPHAEROIDES

Nonlinear polarization spectroscopy in the frequency domain allows rate constant determinations of fast electronic energy and phase relaxations together with characterization of the type of line broadening. Application of this method to the B850 component of the isolated B800–850antenna ofRhodobacter sphaeroides at room temperature shows that B850 is inhomogeneously broadened, with homogeneous widths between 30 and 200 cm−1, depending on the spectral position of the subforms. The corresponding phase relaxation times are clearly in the subpicosecond range. There is also indication of an up-to-now unspecified1–5 ps energy relaxation channel per subunit

Ligand-induced conformational changes in a thermophilic ribose-binding protein

<p>Abstract</p> <p>Background</p> <p>Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments.</p> <p>Results</p> <p>Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile <it>Thermotoga maritima </it>in its ribose-bound and unliganded state. The <it>T. maritima </it>RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (^<it>app</it><it>T</it>_{<it>m </it>}value is 108°C) than the mesophilic <it>Escherichia coli </it>homolog (ecRBP) (^<it>app</it><it>T</it>_{<it>m </it>}value is 56°C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.</p> <p>Conclusion</p> <p>Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.</p

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass

REACTIVITY OF CHLOROPHYLL a/b-PROTEINS AND MICELLAR TRITON X-100 COMPLEXES OF CHLOROPHYLLS a OR b WITH BOROHYDRIDE

The reaction of several plant chlorophyll-protein complexes with NaBH4 has been studied by absorption spectroscopy. In all the complexes studied, chlorophyll b is more reactive than Chi a, due to preferential reaction of its formyl substituent at C-7. The complexes also show large variations in reactivity towards NaBH4 and the order of reactivity is: LHCI > PSII complex > LHCII > PSI > P700 (investigated as a component of PSI). Differential pools of the same type of chlorophyll have been observed in several complexes. Parallel work was undertaken on the reactivity of micellar complexes of chlorophyll a and of chlorophyll b with NaBH4 to study the effect of aggregation state on this reactivity. In these complexes, both chlorophyll a and b show large variations in reactivity in the order monomer > oligomer > polymer with chlorophyll b generally being more reactive than chlorophyll a. It is concluded that aggregation decreases the reactivity of chlorophylls towards NaBH4 in vitro, and may similarly decrease reactivity in naturally-occurring chlorophyll-protein complexes

Noninvasive Instrument-based Tests for Detecting and Measuring Vitreous Inflammation in Uveitis: A Systematic Review

PURPOSE: This systematic review aims to identify instrument-based tests for quantifying vitreous inflammation in uveitis, report the test reliability and the level of correlation with clinician grading. METHODS: Studies describing instrument-based tests for detecting vitreous inflammation were identified by searching bibliographic databases and trials registers. Test reliability measures and level of correlation with clinician vitreous haze grading are extracted. RESULTS: Twelve studies describing ultrasound, optical coherence tomography (OCT), and retinal photography for detecting vitreous inflammation were included: Ultrasound was used for detection of disease features, whereas OCT and retinal photography provided quantifiable measurements. Correlation with clinician grading for OCT was 0.53-0.60 (three studies) and for retinal photography was 0.51 (1 study). Both instruments showed high inter- and intra-observer reliability (>0.70 intraclass correlation and Cohen's kappa), where reported in four studies. CONCLUSION: Retinal photography and OCT are able to detect and measure vitreous inflammation. Both techniques are reliable, automatable, and warrant further evaluation

Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution

AbstractBackground: The study of DNA polymerases in the Pol l family is central to the understanding of DNA replication and repair. DNA polymerases are used in many molecular biology techniques, including PCR, which require a thermostable polymerase. In order to learn about Pol l function and the basis of thermostability, we undertook structural studies of a new thermostable DNA polymerase.Results: A DNA polymerase large, Klenow-like, fragment from a recently identified thermostable strain of Bacillus stearothermophilus (BF) was cloned, sequenced, overexpressed and characterized. Its crystal structure was determined to 2.1 Å resolution by the method of multiple isomorphous replacement.Conclusions: This structure represents the highest resolution view of a Pol l enzyme obtained to date. Comparison of the three Pol l structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure. The polymerase domain is highly conserved in all three proteins. The N-terminal domains are highly divergent in sequence, but retain a common fold. When present, the 3′-5′ proofreading exonuclease activity is associated with this domain. Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements. In BF, these changes result in a blockage of the DNA-binding cleft

Development of a Digital Video-Based Occupational Risk Assessment Method

The development and implementation of an observational video-based risk assessment is described. Occupational risk assessment is one of the most important yet also challenging tasks for employers. Most assessment tools to date use questionnaires, expert interviews, and similar tools. Video analysis is a promising tool for risk assessment, but it needs an objective basis. A video of a plastering worker was recorded using a 360° camera. The recording was then analyzed using the developed observational matrix concerning Work Characteristics, Work Activities as well as potential risks. Risk factors present during the video of the work included lifting, fall from ladder, hazardous substances as well as occasionally bad posture. The worker had no or just one risk factor present during most of the time of the video recording, while only 16 s with more than one risk factor present according to the observational matrix. The paper presents a promising practical method to assess occupational risks on a case-by-case basis. It can help with the risk assessment process in companies which is required by law in some industrialized countries. The matrix in combination with video analysis is a first step toward digital observational risk assessment. It can also be the basis of an automated risk assessment process

Numerical Fracture Analysis Under Temperature Variation by Energetic Method

It is known that temperature change can induce sudden crack propagation especially when the material is composed of fibers. In this fact, the crack growth process under mixed-mode coupling mechanical and thermal loads in orthotropic materials like wood is investigated in this work. The analytical formulation of A integral’s combines the real and virtual mechanical and thermal stress/strain fields under transient diet in 2D. The Mixed Mode Crack Growth specimen providing the decrease of energy release rate during crack propagation is considered in order to compute the various mixed mode ratios. By using three specific routines, the analytical formulation is implemented in finite element software Cast3m. The efficiency of the proposed model is justified by showing the evolution of energy release rate and the stress intensity factors versus crack length and versus temperature variation in time dependent materia