Low frequency dynamics of chymotrypsin by neutron spectroscopy

The low frequency vibrational modes of enzymes have large amplitudes of vibration which should be related to conformational changes that occur during enzyme action. In the present paper we present inelastic neutron scattering measurements for -chymotrypsin that show a peak in the low frequency spectrum. This peak is well defined at 77 K. Gaussian fits yield values of 0.93±0.05, 0.86±0.04, 0.81±0.05, and 0.87±0.06 THz for the peak position at wave vector transfers (Q) of 1.00, 1.40, 1.85, and 3.00 Å^-1, respectively. The full widths at half maximum are all greater than the resolution (0.2 THz) by at least a factor of two. At 298 K a weak peak at about 0.6 THz was observed for Q values of 1.0, 1.4 and 1.85 Å^-1. The data are interpreted in terms of the allowed oscillations of a large globular protein treated as an elastic sphere. Assuming a Raman active oscillation at 0.9 THz it is shown that a peak in the neutron scattering response at 0.6 THz may arise from a rotational shear mode of the chymotrypsin molecule.     

High-resolution waveguide THz spectroscopy of biological molecules

Low-frequency vibrational modes of biological molecules consist of intramolecular modes, which are dependent on the molecule as a whole, as well as intermolecular modes, which arise from hydrogen-bonding interactions and van der Waals forces. Vibrational modes thus contain important information about conformation dynamics of biological molecules, and can also be used for identification purposes. However, conventional Fourier transform infrared spectroscopy and terahertz time-domain spectroscopy (THz-TDS) often result in broad, overlapping features that are difficult to distinguish. The technique of waveguide THz-TDS has been recently developed, resulting in sharper features. For this technique, an ordered polycrystalline film of the molecule is formed on a metal sample plate. This plate is incorporated into a metal parallel-plate waveguide and probed via waveguide THz-TDS. The planar order of the film reduces the inhomogeneous broadening, and cooling of the samples to 77K reduces the homogenous broadening. This combination results in the line-narrowing of THz vibrational modes, in some cases to an unprecedented degree. Here, this technique has been demonstrated with seven small biological molecules, thymine, deoxycytidine, adenosine, D-glucose, tryptophan, glycine, and L-alanine. The successful demonstration of this technique shows the possibilities and promise for future studies of internal vibrational modes of large biological molecules.     

Temperature dependence of protein fluorescence in <Emphasis Type="Italic">Rb. sphaeroides</Emphasis> reaction centers frozen to 80 K in the dark or on the actinic light as the indicator of protein conformational dynamics

The differences in the average fluorescence lifetime (τ_av) of tryptophanyls in photosynthetic reaction center (RC) of the purple bacteria Rb. sphaeroides frozen to 80 K in the dark or on the actinic light was found. This difference disappeared during subsequent heating at the temperatures above 250 K. The computer-based calculation of vibration spectra of the tryptophan molecule was performed. As a result, the normal vibrational modes associated with deformational vibrations of the aromatic ring of the tryptophan molecule were found. These deformational vibrations may be active during the nonradiative transition of the molecule from the excited to the ground state. We assume that the differences in τ_av may be associated with the change in the activity of these vibration modes due to local variations in the microenvironment of tryptophanyls during the light activation.     

The single tryptophan residue of human placental lactogen. Effects of modification and cleavage on biologic activity and protein conformation

In order to define further the chemical features of the human placental lactogen (hPL) molecule responsible for its lactogenic activity, two derivatives of the hormone were prepared by treatment with BNPS-skatole (2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine). At a molar ratio of reagent to hPL of 7:1, a derivative was produced in which the single tryptophan was completely oxidized. At higher ratios, a second derivative was formed in which the peptide chain was cleaved at the tryptophan residue and the two resulting fragments remained bound by the disulfide bond between Cys53 and Cys165. Oxidation of the single tryptophan resulted in reduced immunologic activity, reduced helical content as measured by circular dichroism below 240 nm, and changes in the near-UV circular dichroic spectrum, each indicating a change in the conformation of the hPL molecule. Nevertheless, this derivative retained 20% of its ability to bind to lactogenic receptors and 40 to 50% of its ability to stimulate N-acetyllactosamine synthetase in vitro. Cleavage at the tryptophan was not complete, but the loss of immunologic and biologic activity was equivalent to the degree of cleavage, indicating that the cleaved derivative was completely inactive. In addition, separation of the cleaved fragments from intact hormone followed by recombination did not generate any immunologic or biologic activity. We conclude that the single tryptophan of hPL is not essential for the biologic activity of hPL. It is likely that the reduced activity associated with modification or cleavage at the tryptophan residue is due to changes in the conformation of the molecule.     

Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.     

States of tryptophan residues in castor bean hemagglutinin: the perturbing effects of solvents on tryptophans in hemagglutinin and its complexes as analyzed by UV-difference spectroscopy

The states of tryptophan residues in castor bean hemagglutinin (CBH) were analyzed by solvent perturbation studies employing ultraviolet difference spectroscopy. Eight out of 22 tryptophan residues in CBH were exposed to ethylene glycol and glycerol, suggesting that the remaining 14 tryptophan residues are buried in the interior of the CBH molecule. The fraction of tryptophan residues accessible to the perturbant decreased with increase in the molecular size of the perturbant, and only 2 tryptophan residues were exposed to polyethylene glycol 600. Upon binding with raffinose, 2 tryptophan residues were shielded from the perturbing effect of the solvent, and binding of lactose reduced the number of tryptophan residues accessible to the perturbant by 1 mol per mol of protein. Binding of galactose, however, did not change the accessibility of tryptophan to the perturbant. On the other hand, the accessibility of tyrosine to the perturbant remained unchanged after binding with raffinose and lactose, suggesting that tyrosine is not directly involved in the saccharide binding of CBH. Based on these results, it is proposed that one tryptophan residue at the saccharide-binding site on each B-chain of CBH lies on the surface of the protein molecule and is located at a subsite which is accessible to a glucopyranoside moiety in the lactose molecule or a glycopyranosyl-fructofuranosyl moiety in the raffinose molecule, whereas such a residue is not present at the galactopyranoside-recognition site.     

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.     

Excited-state structure and isomerization dynamics of the retinal chromophore in rhodopsin from resonance Raman intensities.

Resonance Raman excitation profiles have been measured for the bovine visual pigment rhodopsin using excitation wavelengths ranging from 457.9 to 647.1 nm. A complete Franck-Condon analysis of the absorption spectrum and resonance Raman excitation profiles has been performed using an excited-state, time-dependent wavepacket propagation technique. This has enabled us to determine the change in geometry upon electronic excitation of rhodopsin's 11-cis-retinal protonated Schiff base chromophore along 25 normal coordinates. Intense low-frequency Raman lines are observed at 98, 135, 249, 336, and 461 cm-1 whose intensities provide quantitative, mode-specific information about the excited-state torsional deformations that lead to isomerization. The dominant contribution to the width of the absorption band in rhodopsin results from Franck-Condon progressions in the 1,549 cm-1 ethylenic normal mode. The lack of vibronic structure in the absorption spectrum is shown to be caused by extensive progressions in low-frequency torsional modes and a large homogeneous linewidth (170 cm-1 half-width) together with thermal population of low-frequency modes and inhomogeneous site distribution effects. The resonance Raman cross-sections of rhodopsin are unusually weak because the excited-state wavepacket moves rapidly (approximately 35 fs) and permanently away from the Franck-Condon geometry along skeletal stretching and torsional coordinates.     

Polarized luminescence of macromolecules with tryptophan markers. Parameters of high-frequency motion and structure of synthetic polypeptides and proteins

On the basis of the suggested in previous paper [1] model of high-frequency motions of indol group forming a part of tryptophan residue side group parameters of indol groups torsional vibrations in tryptophan-containing synthetic polypeptides and proteins were analysed. Results of conformational analysis and experimental data on polarized luminescence obtained in [2-5] were used.     

Transport of Tryptophan into Brain from the Circulating, Albumin-Bound Pool in Rats and in Rabbits

Tryptophan is the only amino acid in the circulation that is bound by albumin, and previous studies have suggested that the brain tryptophan supply is a function of either the free or the albumin-bound pool of tryptophan in blood. Since the albumin molecule per se does not cross the brain capillary wall, i.e., the blood-brain barrier (BBB), the transport of tryptophan from the circulating albumin-bound pool may involve enhanced dissociation of tryptophan from the albumin binding sites within the cerebral microcirculation. This hypothesis was confirmed in the present studies wherein the dissociation constant (KaD) of albumin binding of tryptophan in the rat or rabbit brain microcirculation was measured in vivo. Brain extraction data for [14C]tryptophan determined with the carotid artery injection technique were fit to the Kety-Renkin-Crone equation modified for protein-bound solute. The KaD of albumin binding in the rat or rabbit brain microcirculation under pentobarbital anesthesia was 1.7 +/- 0.1 and 3.9 +/- 1.0 mM, respectively, as compared to the KD value measured in vitro with equilibrium dialysis, 0.13 +/- 0.03 mM. In contrast, the KaD value of albumin binding of tryptophan in vivo in the rabbit brain microcirculation was reduced by ether anesthesia to a value of 2.1 +/- 0.4 mM. This reduction in the KaD under ether anesthesia was associated with a 2.5-fold increase in cerebral blood flow. In addition, dialyzed rabbit serum caused a statistically significant inhibition in [14C]tryptophan influx during ether, but not pentobarbital, anesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.     

The binding of L-tryptophan to serum albumins in the presence of non-esterified fatty acids.

Bovine, human and rat serum albumins were defatted and palmitic acid, oleic acid and lauric acid added in various molar ratios. The binding of L-tryptophan to these albumins was measured at 20 degrees C in a 0.138 M salt solution at pH 7.4, by using an ultrafiltration technique, and analysed in terms of n, the number of available tryptophan-binding sites per albumin molecule, with apparent association constant, k. 2. n and k were 0.90 and 2.3x10(-4)M(minus-1) respectively for defatted bovine serum albumin and 0.87 and 9.7x10(-3)M(-minus-1) for human albumin. Addition of palmitic acid did not decrease n until the molar ratio, fatty acid/bovine albumin, approached and exceeded 2. The decrease in k was small and progressive. In contrast, lauric caused a marked decrease in n and k at ratios as low as 0.5. A similar distinction between the effects on n of palmitic acid and oleic acid and those of lauric acid was seen for human albumin. k for human albumin was not significantly affected by fatty acids under the conditions studied. 3. It is concluded that primary long-chain fatty acid sites interact only weakly with the tryptophan site on albumin and that inhibition of tryptophan binding occurs when secondary long-chain sites are occupied. Primary medium-chain fatty acid sites are distinct from primary long-chain sites but may be grouped with secondary long-chain sites. 4. The relationship between free and bound tryptophan in samples of rat plasma (Stoner et al., 1975) is discussed in terms of a similar but limited study of rat albumin.     

Conformational aspects of the acid-induced fusion mechanism of influenza virus hemagglutinin. Circular dichroism and fluorescence studies

Circular dichroism and tryptophan fluorescence spectroscopy have been used to investigate the structures of the influenza virus membrane glycoprotein hemagglutinin, acid-treated hemagglutinin, and fragments of hemagglutinin derived by proteolysis. The conformational change in hemagglutinin which occurs at the pH of membrane fusion (pH 5-6) was associated with a significant change of the environment of tyrosine residues, a change in the environment of tryptophan residues, but no changes in secondary structure. Tryptic digestion of the hemagglutinin in its low pH conformation which releases one of the subunit polypeptides (HA1) caused minimal changes in tyrosine and tryptophan environments but a small secondary structural change in HA1. The secondary structure of the remainder of the molecule (HA2) was very similar to that predicted from the known x-ray crystallographic structure of the native molecule. However, fluorescence spectroscopy indicated a tertiary change in structure in the coiled coil of alpha-helices which form the fibrous central stem of the molecule. These results are consistent with a conformational change required for membrane fusion which involves a decrease of HA1/HA1, HA1/HA2 interactions and changes in tertiary structure not accompanied by changes in secondary structure.     

Coupling Between Metamolecular Modes and Lattice Diffraction Modes of Metamaterials in Terahertz Region

Plasmonics - The coupling between plasmonic modes and lattice diffraction modes of circular split-ring resonators in terahertz (THz) region has been analyzed by changing the lattice constants. We...     

Venom exonuclease. IV. Intrinsic fluorescence of the exonuclease from Crotalus adamanteus venom

The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).     

Isoamylamine as the Possible Neuroactive Metabolite of l-Leucine

The states of tryptophan residues in Abrus precatorius agglutinin (APA) were analyzed by chemical modification and solvent perturbation UV-difference spectroscopy. The number of tryptophan residues available for N-bromosuccinimide (NBS) oxidation increased with lowering pH, and 20 out of the 24 tryptophans in APA were modified at pH 3.0, while 2 tryptophans were eventually oxidized at pH 5.0. Modification of tryptophan greatly decreased the binding of APA with saccharides, and only 4% of the hemagglutinating activity was retained after modification of 4 tryptophan residues/molecule. When the modification was done in the presence of lactose or galactose, 2 tryptophan residues/molecule remained unmodified with a retention of a fairly high hemagglutinating activity. The data from solvent perturbation UV-difference spectroscopy indicated that 6 tryptophans were on the surface of the APA molecule, and 4 tryptophan residues/molecule were shielded from the perturbing effect of the solvent upon binding with lactose.

Based on these results, we proposed that in the saccharide-binding site on each B-chain of APA there exists one tryptophan residue directly involved in saccharide binding, and near the binding site there is another tryptophan residue whose state is also changeable upon binding with saccharide.     

Polarized luminescence and mobility of tryptophan residues in polypeptide chains

Polarized luminescence was used to study the mobility of tryptophan residues in polypeptide chains of different chemical composition and structural organization. It has been shown that the luminescence depolarization of tryptophan residues in coillike, helical, and β-structural polypeptide chains is mainly caused by “fast” torsional vibrations and “slow” rotational isomerization of indole groups of tryptophan side chains. The characteristics of these types of motions are practically the same for tryptophan residues included in coillike chains of different chemical structure. Helix–coil transitions in copolymers of glutamic acid and lysine with tryptophan (Glu, Trp) and (Lys, Trp) (where side groups of tryptophan residues weakly interact with the surrounding side groups) do not appreciably change the amplitude of torsional vibrations or rotational isomerization. At the same time, in the helical state of glutamic acid–leucine–tryptophan copolymers (Glu, Leu, Trp) and in the β-structural state of (Lys, Trp) copolymers (where direct interactions of Trp side groups with other side groups are possible), the amplitudes of the torsional vibrations are smaller and the rotational isomerization times larger than in the coil. The transition of (Glu, Leu, Trp) polypeptide chains into a compact state is accompanied by a marked decrease of both “fast” and “slow” intra-molecular mobility and by an increase of the contribution made by the rotation of the macromolecule as a whole, as shown by the decrease of the luminescence polarization.     

Fluorescence of human liver alanine aminopeptidase.

Fluorescence of human liver alanine aminopeptidase has been attributed to tryptophan fluorescence. The fluorescence maximum is at 330 nm, 20 nm lower than that for free tryptophan, suggesting that most of the enzyme tryptophans are in a nonpolar environment and are shielded from solvent. Quenching of enzyme fluorescence by iodide, pyridine, and N-methyl nicotinamide also demonstrates that enzyme tryptophan residues are largely buried and inaccessible to solvent. Those accessible are in negatively charged environments. 8-(1'-dimethylaminonaphthalene-5'-sulfonylamido-octanoic acid (8-DNS-octanoic acid) and epsilon-DNS-L-Lys inhibit aminopeptidase. One molecule of inhibitor when bound to the enzyme quenched 57% and 63% of enzyme fluorescence, respectively. Such efficient quenching may indicate a degree of segregation of tryptophan toward the active center.     

Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC.

The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300-380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380-500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate.The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL(S146A). Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments.As a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids.