Biomedical subjects

A A Yee

Publications and source records attributed to A A Yee.

5 recordsLinked to original sources

PubMed2001

The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold.

Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.

Amino Acid Sequence↗

PubMed1998

Quantitative hydroxyl radical footprinting reveals cooperative interactions between DNA-binding subdomains of PU.1 and IRF4.

Quantitative hydroxyl radical footprinting and fluorescence polarization measurements have been used to determine the dissociation constants (Kd) of complexes between the ets domain of the murine transcription factor PU.1 and three different DNA fragments. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Iglambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex. With the use of quantitative hydroxyl radical footprinting we obtained binding isotherms for individual protected nucleotides and contact sites on both strands of the DNA. Kd values of (1.53 +/- 0. 12) x 10(-)8 M were found for the lambdaB element, (3.60 +/- 0.65) x 10(-)8 M for the SV40 enhancer site, and (2.28 +/- 0.27) x 10(-)8 M for the sequence used in the crystal structure. In addition, the binding of a second protein, the DNA binding domain of IRF4, to the lambdaB site by itself and in the presence of PU.1 was analyzed. The IRF4 DBD shows three footprints on the TTCC strand and one footprint on the GGAA strand of the lambdaB element. The dissociation constant for the binary IRF4 DBD-lambdaB complex equals (5.59 +/- 0.60) x 10(-)7 M. The Kd value of the IRF4-lambdaB interaction is reduced by a factor of 5 in the presence of two different DNA-bound PU.1 protein constructs, PU.1 DBD and a PU.1 construct containing the PEST domain (PU.1-PEST). A similar decrease of the Kd value was observed for the binding of PU.1-PEST in the presence of DNA-bound IRF4 DBD demonstrating a cooperative interaction between the PU. 1-PEST and IRF4 DBD. On the basis of the hydroxyl radical footprints in the ternary PU.1/IRF4/lambdaB complex, a model for the interactions between the two proteins and the lambdaB site was developed. The DNA binding domains of both proteins bind the DNA in the major groove with potential protein-protein interactions near the intervening minor groove.

Animals↗

PubMed1998

Cooperative interaction between the DNA-binding domains of PU.1 and IRF4.

The two lymphoid-specific transcription factors PU.1 and IRF4 form a cooperative ternary complex at immunoglobulin enhancer elements such as the lambdaB and kappaE3' sites. We report here that the synergy of this interaction can be reconstituted in part with the DNA-binding domains of the two proteins. The minimal DNA binding-domain of IRF4 was mapped to residues 20 to 137, corresponding to the conserved DNA-binding region of other interferon regulatory factors (IRFs). This domain can bind weakly to a synthetic murine lambdaB element, while IRF4 constructs that contain residues 1 to 19 require the presence of PU.1 for DNA-binding at similar concentrations. Fluorescence polarization of fluorescein-labelled DNA was used to show that the presence of residues 1 to 19 decreases the binding affinity of IRF4 N-terminal constructs from two- to fivefold. However, all constructs bound better to the lambdaB element in the presence of the DNA-binding domain of PU.1. This cooperative interaction was not dependent on phosphorylation of the PEST domain of PU.1, but was dependent on the proper spacing of the binding sites for PU.1 and IRF4. These data suggest that at least part of the cooperative interaction between full-length PU.1 and IRF4 involves the DNA-binding domains of the two proteins. NMR spectroscopy of 15N-labelled PU.1 and IRF4 constructs indicates that the PEST domain of PU.1 and residues 1 to 19 of IRF4 may be unstructured in the isolated proteins.

Amino Acid Sequence↗

PubMed1997

The interactions with solvent, heat stability, and 13C-labelling of alamethicin, an ion-channel-forming peptide.

The peptide alamethicin was labelled with 13C and 15N by growing the fungus Trichoderma viride in a medium containing [U-13C] glucose and K15NO3. Spin-echo difference spectroscopy showed that 13C was incorporated to a level of about 50% and 15N to about 98%. Incorporation of 13C into the peptide provided residue-specific probes of the interactions with solvent and heat stability of this ion-channel-forming peptide. All of the carbonyl carbons and the alpha-carbons of the alpha-aminoisobutyric acid [Ala(Me)] residues of alamethicin in methanol were assigned using two-dimensional and three-dimensional heteronuclear correlation experiments. Measurements of 1JC'N revealed hydrogen bonding with solvent at residues 1 and 19 at the ends of the peptide and at Gly11 in the middle. The data also support the thesis [see Juranic, N., Ilich, P. K. & Macara, S. (1995) J. Am. Chem. Soc. 117, 405-410 that intramolecular hydrogen bonds in proteins and peptides are weaker than hydrogen bonds to solvent. The sensitivity of alamethicin carbonyl and proton chemical shifts to perturbation by dimethyl sulfoxide correlates well with the calculated solvent accessibilities of the carbonyls in the crystal structures and reveals residues in the middle of the peptide and at the C-terminus which interact with solvent. Taken together with the 1JC'N measurements, the data support a model in which hydrogen bonding to solvent at the Gly11/Leu12 amide could provide a site of hydration in the interior of the alamethicin channel structure. The temperature dependencies of the carbonyl chemical shifts support the suggestion that the peptide is flexible in the regions where solvent interacts with the backbone of the peptide. The linear temperature dependence of the carbonyl chemical shifts and molar ellipticity indicate that, due to steric constraints at the Ala(Me) residues, the peptide folding/unfolding transition is non-cooperative and that the peptide is remarkably heat stable.

Alamethicin↗

PubMed1992

Uniform 15N labeling of a fungal peptide: the structure and dynamics of an alamethicin by 15N and 1H NMR spectroscopy.

An alamethicin, secreted by the fungus Trichoderma viride and containing a glutamine at position 18 instead of the usual glutamic acid, has been uniformly labeled with 15N and purified by HPLC. The extent of 15N incorporation at individual backbone and side-chain sites was found to vary from 85% to 92%, as measured by spin-echo difference spectroscopy. The proton NMR spectrum of the peptide dissolved in methanol was assigned using correlation spectroscopies and nuclear Overhauser enhancements (NOE) measured in the rotating frame. The 15N resonances were assigned by the 2D 1H-15N correlation via heteronuclear multiple-quantum coherence experiment. NOEs and 3JNHC alpha H coupling constants strongly suggest that, in methanol, from Aib-3 to Gly-11, the peptide adopts a predominantly helical conformation, in agreement with previous 1H NMR studies [Esposito, G., Carver, J.A, Boyd, J., & Campbell, I.D. (1987) Biochemistry 26, 1043-1050; Banerjee, U., Tsui, F.-P., Balasubramanian, T.N., Marshall, G.R., & Chan, S I. (1983) J. Mol. Biol. 165, 757-775]. The conformation of the carboxyl terminus (12-20) is less well determined, partly because the amino acid composition reduces the number of NOEs and coupling constants which can be determined by 1H NMR spectroscopy. The 3JNHC alpha H in the C-terminus suggest the possibility of conformational averaging at Leu-12, Val-15, and Gln-19, an interpretation which is supported by a recent molecular dynamics simulation of the peptide [Fraternalli, F. (1990) Biopolymers 30, 1083-1099].(ABSTRACT TRUNCATED AT 250 WORDS)

Alamethicin↗