Ultrafast quenching of tryptophan fluorescence in
proteins: Interresidue and intrahelical electron
transfer
Weihong Qiu
Quenching of tryptophan fluorescence in proteins has been critical to
the understanding of protein dynamics and enzyme reactions using
tryptophan as a molecular optical probe. We report here our systematic
examinations of potential quenching residues with more than 40
proteins. With site-directed mutation, we placed tryptophan to desired
positions or altered its neighboring residues to screen quenching
groups among twenty amino acid residues and of peptide backbones. With
femtosecond resolution, we observed the ultrafast quenching dynamics
within 100 ps and identified two ultrafast quenching groups, the
carbonyl- and sulfur-containing residues. The former is glutamine and
glutamate residues and the later is disulfide bond and cysteine
residue. The quenching by the peptide-bond carbonyl group as well as
other potential residues mostly occurs in longer than 100 ps. These
ultrafast quenching dynamics occur at van der Waals distances through
intraprotein electron transfer with high directionality. Following
optimal molecular orbital overlap, electron jumps from the benzene
ring of the indole moiety in a vertical orientation to the LUMO of
acceptor quenching residues. Molecular dynamics simulations were
invoked to elucidate various correlations of quenching dynamics with
separation distances, relative orientations, local fluctuations and
reaction heterogeneity. These unique ultrafast quenching pairs, as
recently found to extensively occur in high-resolution protein
structures, may have significant biological implications.
Simple is beautiful: a straightforward approach to improve
the delineation of true and false positives in PSI-BLAST
searches
Marianne Lee
The deluge of biological information from different genomic initiatives and the rapid advancement in biotechnologies have made bioinformatics tools an integral part of modern biology. One of the most widely used techniques is sequence alignment. Among all sequence comparison tools, BLAST and PSI-BLAST are probably the most popular in the field. PSI-BLAST, which uses a profile (PSSM) built from families of related sequences and an iterative search strategy, is more sensitive than BLAST in detecting weak homologies, thus making it suitable for searching remote homologs. Many refinements have been made to improve PSI-BLAST since its first launch, and its computational efficiency and high specificity have been much touted. Nevertheless, corruption of its profile due to the incorporation of false positive sequences remains a major challenge. In this paper, we propose a simple and elegant approach to resolve this problem. Our hypothesis is that combining results from the first (least-corrupted) profile with results from later (most sensitive) iterations of PSI-BLAST provides a better discriminator for true and false hits. Accordingly, we have derived a formula that utilizes the E-values from these two PSI-BLAST iterations to obtain a figure of merit for rank-ordering the hits. Our verification results based on a "gold-standard" test set of 103 queries indicate that this figure of merit does indeed delineate true positives from false positives better than PSI-BLAST E-values. Perhaps what is most notable about this strategy is that it is simple and straightforward to implement.