Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence

Axelsen, Paul H.; Prendergast, Franklyn G.

doi:10.1016/s0006-3495(89)82651-7

Cited by 42 publications

(32 citation statements)

References 53 publications

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“…From these analyses we conclude that significant indole motions with correlation times for 2 to 15 ps are not consistent with the present data. Recently, two divergent reports appeared on simulated molecular dynamics of the trp residue in RNase T 1 (Axelsen and Prendergast 1989;MacKerrel et al 1988). Our experimental results on RNase T1 are not in agreement with the 100ps-22 degree motion found by MacKerrel et al (1988), but are consistent with the simulated anisotropy plateau of 0.279 found by Axelsen and Prendergast (1989).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, two divergent reports appeared on simulated molecular dynamics of the trp residue in RNase T 1 (Axelsen and Prendergast 1989;MacKerrel et al 1988). Our experimental results on RNase T1 are not in agreement with the 100ps-22 degree motion found by MacKerrel et al (1988), but are consistent with the simulated anisotropy plateau of 0.279 found by Axelsen and Prendergast (1989). It should be emphasized that a rigorous comparison of experimental results and calculations requires that results be compared for the same proteins.…”

Section: Discussionmentioning

confidence: 99%

“…1985;Lakowicz etal. 1986a;Eftink et al 1989;Eftink and Ghiron 1987a), and in the case of RNase T1 the subject of molecular dynamics simulations (Axelsen and Prendergast 1989;Mackerell et al 1989).…”

Section: Introductionmentioning

confidence: 99%

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Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

et al. 1991

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We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T1, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40 degrees C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T1, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

et al. 1991

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“…In the case of azurin, mutagenesis of the two hydrophobic residues I7 and F110 to serine (located in the vicinity of W48) leads to a red shift of the emission maximum [39,40]. Apart from this study, W59 in RNase T 1 is probably the only other example (to our knowledge) of a tryptophan, localized in an environment containing polar residues and ordered water molecules, emitting at a low‐emission wavelength of 322 nm [25,41].…”

Section: Fluorescence Propertiesmentioning

confidence: 99%

Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single‐tryptophan mutants

Pattanaik

Ravindra

Sengupta

et al. 2003

European Journal of Biochemistry

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Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blueshifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-Å crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid.

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“…27,28 Axelsen and co-workers have compared the difference between fluorescence decay of Trp59 in RNT1 with molecular dynamic simulations. [29][30][31] We have previously studied the relative free energy difference in binding of 3′GMP and 2-aminopurine-3′-monophosphate. 32,33 In this study we have investigated the structure of the ligand 3′GMP binding to RNT1.…”

Section: Introductionmentioning

confidence: 99%

A 1.2 ns Molecular Dynamics Simulation of the Ribonuclease T₁−3‘-Guanosine Monophosphate Complex

Elofsson

Nilsson

1996

J. Phys. Chem.

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In this study a molecular dynamics simulation of ribonuclease T 1 in complex with its product 3′GMP has been performed for a time span of 1.2 ns. By increasing the simulation time, the dynamic properties of the protein could be analyzed in more detail. Water has been included in an 18 Å radius sphere around the active site. We find that in this system 600 ps of simulation is needed before a stable conformation is reached as measured by the RMS (root mean square) difference from the starting structure, which was adapted from the crystallographic structure of ribonuclease T 1 (RNT1)-2′GMP. The increased length of the simulation gives new opportunities to study the behavior of protein motion. Several parameters such as the temperature factors and correlation functions change noticeably during the simulation. A possible mechanism for the diffusion of guanine from or to the active site was detected. This involves a two-step procedure, in which Tyr45 first opens the binding pocket, whereupon the phosphate rearranges its contacts with the catalytic residues. The motion of Tyr45 is different from what has been proposed from the structure of nucleotidefree RNT1. The binding site closes again during the simulation. We suggest that this mechanism is used to remove the product after catalysis, but not necessarily for the association of the substrate. These rearrangements are much slower than could have been detected in a shorter simulation.

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Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence

Cited by 42 publications

References 53 publications

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single‐tryptophan mutants

A 1.2 ns Molecular Dynamics Simulation of the Ribonuclease T₁−3‘-Guanosine Monophosphate Complex

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Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence

Cited by 42 publications

References 53 publications

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single‐tryptophan mutants

A 1.2 ns Molecular Dynamics Simulation of the Ribonuclease T1−3‘-Guanosine Monophosphate Complex

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A 1.2 ns Molecular Dynamics Simulation of the Ribonuclease T₁−3‘-Guanosine Monophosphate Complex