As for many enzymes, the enzymatic pathway of triosephosphate isomerase (TIM) includes the partially rate determining motion of an active site loop (loop 6, residues 166-176), which must remain closed during chemistry but must open just before product release. The motion of this loop was monitored using laser induced temperature-jump relaxation spectroscopy at nanosecond to millisecond time resolution. Trp168 in the hinge of the mobile loop served as a fluorophore reporter in a mutant of the yeast enzyme. The opening rate was studied as a function of the concentration of glycerol 3-phosphate, a substrate surrogate. Monoexponential kinetics were observed; assuming a simple two-step ligand release mechanism involving an encounter complex intermediate, the time scales of loop opening and closing were derived. The opening rate of the loop at 25 degrees C was determined to be 2500 +/- 1000 s(-1), in remarkable agreement with solution and solid state NMR measurements. The closing rate at the same temperature was 46,700 +/- 1800 s(-1). The rates were also studied as a function of the sample temperature following the jump. Enthalpies of activation of the loop motion, DeltaH(close) and DeltaH(open), were estimated to be 13.8 and 14.1 kcal/mol, respectively. The enthalpy of dissociation estimated from the kinetic studies is in reasonable agreement with steady-state values. Moreover, the enthalpy was dissected, for the first time, into components associated with ion binding and with protein conformational change. The enthalpy of the release reaction appeared to have a substantial contribution from the dissociation of the ligand from the encounter complex, found to be endothermic at 6 kcal/mol. In contrast, the population ratio of the open to closed loop conformations is found to favor the closed conformation but to be substantially less temperature dependent than the release step. Preliminary data of other ligands show that G3P behavior resembles that of the substrate but differs from 2-phosphoglycolate, a tight binding inhibitor, and phosphate. This study represents one of the first detailed comparisons between NMR and fluorescence based probes of protein motion and results in good agreement between the methods. The data in aggregate support a model in which the rate of the loop opening for TIM is dependent on the ligand and results in opening rates in the presence of the product that are comparable to enzymatic throughput, kcat.

The spectroscopic properties of spheroidene and a series of spheroidene analogs with extents of π-electron conjugation ranging from 7 to 13 carbon−carbon double bonds were studied using steady-state absorption, fluorescence, fluorescence excitation, and time-resolved absorption spectroscopy. The spheroidene analogs studied here were 5‘,6‘-dihydro-7‘,8‘-didehydrospheroidene, 7‘,8‘-didehydrospheroidene, and 1‘,2‘-dihydro-3‘,4‘,7‘,8‘-tetradehydrospheroidene and taken together with data from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene already published (DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J.; Farhoosh, R.; Frank, H. A. Biochim. Biophys. Acta 1992, 1102, 107) provide a systematic series of molecules for understanding the molecular features that control energy transfer to bacteriochlorophyll in photosynthetic bacterial light-harvesting complexes. All of the molecules were purified by high-pressure liquid chromatographic techniques prior to the spectroscopic experiments. The absorption spectra of the molecules were observed to red-shift with increasing extent of π-electron conjugation. The room temperature fluorescence data show a systematic crossover from dominant S1 → S0 (2Ag → 11Ag) emission to dominant S2 → S0 (11Bu → 11Ag) with increasing extent of conjugation. The S2 fluorescence quantum yields of all the carotenoids in the series were measured here and indicate that 3,4-dihydrospheroidene with nine carbon−carbon double bonds has an S2 quantum yield of (2.7 ± 0.3) × 10-4 which is the highest value in the series. The lifetimes of the S1 states of the molecules were determined from time-resolved transient absorption spectroscopy and found to decrease as the conjugated chain length increases. The transient data are discussed in terms of the energy gap law for radiationless transitions which allows a prediction of the S1 energies of the molecules. The implications of these results for the process of light harvesting by carotenoids in photosynthesis are discussed.

Spheroidene and a series of spheroidene analogues with extents of π-electron conjugation ranging from 7 to 13 carbon−carbon double bonds were incorporated into the B850 light-harvesting complex of Rhodobacter sphaeroides R-26.1. The structures and spectroscopic properties of the carotenoids and the dynamics of energy transfer from the carotenoid to bacteriochlorophyll (BChl) in the B850 complex were studied by using steady-state absorption, fluorescence, fluorescence excitation, resonance Raman, and time-resolved absorption spectroscopy. The spheroidene analogues used in this study were 5‘,6‘-dihydro-7‘,8‘-didehydrospheroidene, 7‘,8‘-didehydrospheroidene, and 1‘,2‘-dihydro-3‘,4‘,7‘,8‘-tetradehydrospheroidene. These data, taken together with results from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene, and spheroidene already published (Frank, H. A.; Farhoosh, R.; Aldema, M. L.; DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J. Photochem. Photobiol. 1993, 57, 49. Farhoosh, R.; Chynwat, V.; Gebhard, R.; Lugtenburg, J.; Frank, H. A. Photosynth. Res. 1994, 42, 157), provide a systematic series of molecules for understanding the molecular features that determine the mechanism of energy transfer from carotenoids to BChl in photosynthetic bacterial light-harvesting complexes. The data support the hypothesis that only carotenoids having 10 or less carbon−carbon double bonds transfer energy via their 2Ag (S1) states to BChl to any significant degree. Energy transfer via the 11Bu (S2) state of the carotenoid becomes more important than the S1 route as the number of conjugated carbon−carbon double bonds increases. The results also suggest that the S2 state associated with the Q x transition of the B850 BChl is the most likely acceptor state for energy transfer originating from both the 21Ag (S1) and 11Bu (S2) states of all carotenoids.

Acta Technologica Agriculturae 1/2016Dušan Páleš et al.The most effective way for determination of curves for practical use is to use a set of control points. These control points can be accompanied by other restriction for the curve, for example boundary conditions or conditions for curve continuity (Sederberg, 2012). When a smooth curve runs only through some control points, we refer to curve approximation. The B-spline curve is one of such approximation curves and is addressed in this contribution. A special case of the B-spline curve is the Bézier curve Rédl et al., 2014). The B-spline curve is applied to a set of control points in a space, which were obtained by measurement of real vehicle movement on a slope (Rédl, 2007(Rédl, , 2008. Data were processed into the resulting trajectory (Rédl, 2012;Rédl and Kučera, 2008). Except for this, the movement of the vehicle was simulated using motion equations (Rédl, 2003;Rédl and Kročko, 2007). B-spline basis functionsBézier basis functions known as Bernstein polynomials are used in a formula as a weighting function for parametric representation of the curve (Shene, 2014). B-spline basis functions are applied similarly, although they are more complicated. They have two different properties in comparison with Bézier basis functions and these are: 1) solitary curve is divided by knots, 2) basis functions are not nonzero on the whole area. Every B-spline basis function is nonzero only on several neighbouring subintervals and thereby it is changed only locally, so the change of one control point influences only the near region around it and not the whole curve.These numbers are called knots, the set U is called the knot vector, and the half-opened interval 〈u i , u i + 1 ) is the i-th knot span. Seeing that knots u i may be equal, some knot spans may not exist, thus they are zero. If the knot u i appears p times, hence u i = u i + 1 = ... = u i + p -1 , where p >1, u i is a multiple knot of multiplicity p, written as u i (p). If u i is only a solitary knot, it is also called a simple knot. If the knots are equally spaced, i.e. (u i + 1 -u i ) = constant, for every 0 ≤ i ≤ (m -1), the knot vector or knot sequence is said uniform, otherwise it is non-uniform.Knots can be considered as division points that subdivide the interval 〈u 0 , u m 〉 into knot spans. All B-spline basis functions are supposed to have their domain on 〈u 0 , u m 〉. We will use u 0 = 0 and u m = 1.To define B-spline basis functions, we need one more parameter k, which gives the degree of these basis functions. Recursive formula is defined as follows:This definition is usually referred to as the Cox-de Boor recursion formula. If the degree is zero, i.e. k = 0, these basis functions are all step functions that follows from Eq. (1). N i, 0 (u) = 1 is only in the i-th knot span 〈u i , u i + 1 ). For example, if we have four knots u 0 = 0, u 1 = 1, u 2 = 2 and u 3 = 3, knot spans 0, 1 and 2 are 〈0, 1), 〈1, 2) and 〈2, 3), and the basis functions of degree 0 are N 0, 0 (u) = 1 on interval 〈0, 1) Acta In this co...

A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C2=O band of bound substrate mimic and the C4-H stretch of NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong ‘anchor’ within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme.

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of hIAPP22–29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman, and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22–29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β-sheet secondary structure. TEM images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the -C=C- ring mode (ca. 1600 cm−1) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22–29.

The correlation of the UVRR νW3 mode with the tryptophan χ 2,1 dihedral angle (1-3) has been extended to a full, 360° rotation. The three-fold periodicity of the relationship (cos 3χ 2,1 ) over 360°r esults in up to six dihedral angles for a given νW3. Consideration of a Newman plot of dihedral angles for proteinaceous tryptophans taken from the Protein Data Bank shows that sterically hindered ranges of dihedral angle reduce the possible χ 2,1 to one or two. However, not all proteinaceous tryptophans follow the νW3-χ 2,1 relationship. Hydrogen bonding at the indole amine, weaker, electrostatic cation-π and anion quadrapole interactions and environmental hydrophobicity are examined as possible contributing factors to noncompliance with the relationship. This evaluation suggests that cumulative weak electrostatic and nonpolar interactions characterize the environment of tryptophans that obey the νW3-χ 2,1 relationship, matching that of the crystalline tryptophan derivatives used to derive the relationship. In the absence of methods to quantify these weak interactions, measurement of the full width half maximum bandwidth (FWHM) of the W3 band is suggested as a primary screen for evaluating the applicability of the νW3 -χ 2,1 relationship. Keywords tryptophan; protein structure; triose phosphate isomerase; DFT calculations; resonance Raman spectroscopy; dihedral angle; UVRR The utility of spectroscopy with respect to protein structure lies in its predictive value in the absence of crystallographic structure. In UV resonance Raman spectroscopy, tryptophan residues are structural bellwethers because they can be selectively probed at 229 nm. Tryptophan vibrational band are good markers for hydrophobic interaction (3-6), hydrogen

Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophylic lactate dehydrogenase, bsLDH•NADH•oxamate, were studied with site specific resolution by laser induced temperature jump relaxation spectroscopy having a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions at different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the ms - μs time-scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called `loop motion' is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on catalytic mechanism of bsLDH is discussed.