Linear dichroism amplification: Adapting a long-known technique for ultrasensitive femtosecond IR spectroscopy

We demonstrate strong amplification of polarization-sensitive transient IR signals using a pseudo-null crossed polarizer technique first proposed by Keston and Lospalluto [Fed. Proc. 10, 207 (1951)] and applied for nanosecond flash photolysis in the visible by Che et al. [Chem. Phys. Lett. 224, 145 (1994)]. We adapted the technique to ultrafast pulsed laser spectroscopy in the infrared using photoelastic modulators, which allow us to measure amplified linear dichroism at kilohertz repetition rates. The method was applied to a photoswitch of the N-alkylated Schiff base family in order to demonstrate its potential of strongly enhancing sensitivity and signal to noise in ultrafast transient IR experiments, to simplify spectra and to determine intramolecular transition dipole orientations

Unlocking the Double Bond in Protonated Schiff Bases by Coherent Superposition of S_{1} and S_{2}

The primary event occurring during the E-to-Z photoisomerization reaction of retinal protonated Schiff base (rPSB) is single-to-double bond inversion. In this work we examine the nuclear dynamics that occurs when the initial excited state is a superposition of the S_{1} and S_{2} electronic excited states that might be created in a laser experiment. The nuclear dynamics is dominated by double bond inversion that is parallel to the derivative coupling vector of S_{1} and S_{2}. Thus, the molecule behaves as if it were at a conical intersection even if the states are nondegenerate

MTHFR C677T polymorphism analysis: A simple, effective restriction enzyme-based method improving previous protocols

Background: 5,10-Methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is one of the most studied genetic variations in the human genome. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the most used techniques to characterize the point mutations in genomic sequences because of its suitability and low cost. The most widely used method for the MTHFR C677T polymorphism characterization was developed by Frosst et al. (1995) but appears to have some technical limitations. The aim of this study was to propose a novel PCR-RFLP method for the detection of this polymorphism. Methods: In order to retrieve all published articles possibly describing any PCR-RFLP methods useful to analyze MTHFR C677T polymorphism, we performed systematic queries on PubMed, using a combination of Boolean operators (AND/OR) and MeSH terms. Amplify software was used in order to design a new primer pair following the optimal standard criteria. Primer-BLAST software was used to check primer pair's biological specificity. Results: The analysis of previous literature showed that PCR-RFLP method remains the most used technique. None of the 108 primer pairs described was ideal with regard to main accepted primer pair biochemical technical parameters. The new primer pair amplifies a DNA-fragment of 513 base pair (bp) that, in the presence of the polymorphism, is cut by Hinf I enzyme in two pieces of 146 bp and 367 bp and clearly visible on 2% agarose gel. The level of expertise and the materials required are minimal and the protocol takes one day to carry out. Conclusion: Our original PCR-RFLP strategy, specifically designed to make the analysis optimal with respect to PCR primers and gel analysis, fits the ideal criteria compared to the widely used strategy by Frosst et al (1995) as well as any other PCR-RFLP strategies proposed for MTHFR C677T polymorphism genotyping to date

Toward An Understanding Of The Retinal Chromophore In Rhodopsin Mimics

Recently, a rhodopsin protein mimic was constructed by combining mutants of the cellular retinoic acid binding protein II (CRABPII) with an all-trans retinal chromophore. Here, we present a combine computational quantum mechanics/molecular mechanics (QM/MM) and experimental ultrafast kinetic study of CRABPII. We employ the QM/MM models to study the absorption (lambda(a)(max)), fluorescence (lambda(f)(max)), and reactivity of a CRABPII triple mutant incorporating the all-trans protonated chromophore (PSB-KLE-CRABPII). We also study the spectroscopy of the same mutant incorporating the unprotonated chromophore and of another double mutant incorporating the neutral unbound retinal molecule held inside the pocket. Finally, for PSB-KLE-CRABPII, stationary fluorescence spectroscopy and ultrafast transient absorption spectroscopy resolved two different evolving excited state populations which were computationally assigned to distinct locally excited and charge-transfer species. This last species is shown to evolve along reaction paths describing a facile isomerization of the biologically relevant 11-cis and 13-cis double bonds. This work represents a first exploratory attempt to model and study these artificial protein systems. It also indicates directions for improving the QM/MM models so that they could be more effectively used to assist the bottom-up design of genetically encodable probes and actuators employing the retinal chromophore

A Comparative Study of Modern Homology Modeling Algorithms for Rhodopsin Structure Prediction

Rhodopsins are seven α-helical membrane proteins that are of great importance in chemistry, biology, and modern biotechnology. Any in silico study on rhodopsin properties and functioning requires a high-quality three-dimensional structure. Due to particular difficulties with obtaining membrane protein structures from the experiment, in silico prediction of the three-dimensional rhodopsin structure based only on its primary sequence is an especially important task. For the last few years, significant progress was made in the field of protein structure prediction, especially for methods based on comparative modeling. However, the majority of this progress was made for soluble proteins and further investigations are needed to achieve similar progress for membrane proteins. In this paper, we evaluate the performance of modern protein structure prediction methodologies (implemented in the Medeller, I-TASSER, and Rosetta packages) for their ability to predict rhodopsin structures. Three widely used methodologies were considered: two general methodologies that are commonly applied to soluble proteins and a methodology that uses constraints that are specific for membrane proteins. The test pool consisted of 36 target-template pairs with different sequence similarities that was constructed on the basis of 24 experimental rhodopsin structures taken from the RCSB database. As a result, we showed that all three considered methodologies allow obtaining rhodopsin structures with the quality that is close to the crystallographic one (root mean square deviation (RMSD) of the predicted structure from the corresponding X-ray structure up to 1.5 Å) if the target-template sequence identity is higher than 40%. Moreover, all considered methodologies provided structures of average quality (RMSD < 4.0 Å) if the target-template sequence identity is higher than 20%. Such structures can be subsequently used for further investigation of molecular mechanisms of protein functioning and for the development of modern protein-based biotechnologies