Insensitivity of Tryptophan Fluorescence to Local Charge Mutations

Abstract

The steady state fluorescence spectral maximum (λmax) for tryptophan 140 of Staphylococcal nuclease remains virtually unchanged when nearby charged groups are removed by mutation, even though large electrostatic effects on λmax might be expected. To help understand the underlying mechanism of this curious result, we have modeled λmax with three sets of 50-ns molecular dynamics simulations in explicit water, equilibrated with excited state and with ground state charges. Semiempirical quantum mechanics and independent electrostatic analysis for the wild-type protein and four charge-altering mutants were performed on the chromophore using the coordinates from the simulations. Electrostatic contributions from the nearby charged lysines by themselves contribute 30-90 nm red shifts relative to the gas phase, but in each case, contributions from water create compensating blue shifts that bring the predicted λmax within 2 nm of the experimental value, 332 ± 0.5 nm for all five proteins. Although long-range collective interactions from ordered water make large blue shifts, crucial for determining the steady state λmax for absorption and fluorescence, such blue shifts do not contribute to the amplitude of the time dependent Stokes shift following excitation, which comes from nearby charges and only ∼6 waters tightly networked with those charges. We therefore conclude that for STNase, water and protein effects on the Stokes shift are not separable.

Molecular Mechanism of Wide Photoabsorption Spectral Shifts of Color Variants of Human Cellular Retinol Binding Protein II

“…The large dielectric compensation from bulk water molecules was also found in a previous study by Scott and Callis. 23 The observed screening of bulk water molecule is consistent with the experimentally observed insensitivity of polarity of residues replacing arginine at the position of 58 described above. Moreover, the contribution is largely compensated by an accumulated contribution of many amino acids in the C-terminal half of the protein, which form a β-sheet secondary structure next to PSB half of the chromophore ( Figure S10), and consequently, the overall contribution of the protein comes to be −3.8 kcal/mol, which is smaller than that of the water molecules near PSB, −6.9 kcal/ mol ( Table 1).…”

Charge Invariant Protein–Water Relaxation in GB1 via Ultrafast Tryptophan Fluorescence

“…Water decreases its redshifting influence a full 3500 cm –1 at pH 5 depending on whether Glu27 or Glu42 is protonated, yet protein contributions almost completely compensate that. Exactly the same behavior was found in QM-MM simulations, 50 of the local charge-mutated SNase experiments by Qiu et al; 30 in these and several other cases, 15,23−26 a delicate balance between water and protein make the steady state reaction field response to Trp excitation an invariant (Figures S4 and S5, Supporting Information, show dynamics of this compensation).…”

Insight into the fluorescence quenching of Trp214 at HSA by the Dimetridazole ligand from simulation

“…As noted previously [17], the dipole moment of the 1 La state and the interaction of this state with polar solvents are enhanced by internal CT between the conjugated five-membered pyrrole and six-membered benzene rings, whereas 1 Lb arises mainly from electron redistribution within the benzene unit. The shift in the electron density (ED) from the pyrrole ring to the benzene ring upon excitation to the 1 La state is very sensitive to the environment due to the involvement of the polar nitrogen atom in this transition, as have been confirmed by numerous experimental studies and quantum-mechanical computations [3,53,57].…”

“…These calculated point charges were kept fixed during the MD simulations because the total molecular charge distribution dominates the interaction with other surrounding particles at the VdW distances important for the complexes rather than any minor charge changes on the individual atoms. It has for instance been shown that feeding the QM-calculated point charges back to the MD simulation only lead to minor improvements in the description of the interaction of the indole chromophore with the microenvironment [53].…”