Fuzzy oil drop model to interpret the structure of antifreeze proteins and their mutants

Mutations in proteins introduce structural changes and influence biological activity: the specific effects depend on the location of the mutation. The simple method proposed in the present paper is based on a two-step model of in silico protein folding. The structure of the first intermediate is assumed to be determined solely by backbone conformation. The structure of the second one is assumed to be determined by the presence of a hydrophobic center. The comparable structural analysis of the set of mutants is performed to identify the mutant-induced structural changes. The changes of the hydrophobic core organization measured by the divergence entropy allows quantitative comparison estimating the relative structural changes upon mutation. The set of antifreeze proteins, which appeared to represent the hydrophobic core structure accordant with “fuzzy oil drop” model was selected for analysis

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand–protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand–protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems

13CHD2 methyl group probes of millisecond time scale exchange in proteins by 1H relaxation dispersion: an application to proteasome gating residue dynamics.

A pulse scheme is presented for quantifying millisecond time scale chemical exchange processes in proteins by measuring (1)H CPMG relaxation dispersion profiles of (13)CHD(2) methyl groups. The use of (13)CHD(2) isotopomers for (1)H methyl dispersion experiments eliminates problems with interconversion between differentially relaxing proton transitions that complicate the extraction of accurate exchange parameters when (13)CH(3) probes are used. Good agreement is demonstrated between extracted chemical shift differences from fits of dispersion profiles and the corresponding differences measured independently on a model exchanging system, validating the experiment. The methodology is applied to the gating residues of the T. acidiphilium proteasome that are shown to undergo extensive motion on the millisecond time scale

Structural biology: analysis of `downhill` protein folding

There is controversy as to whether homologues from the peripheral subunit binding domain family of small proteins fold `downhill` (that is, non-cooperatively, in the absence of free-energy barriers between conformations) and whether they modulate their size for biological function. Sadqi et al. claim that Naf-BBL-a naphthylalanine-labelled, truncated version of this domain-folds in this way, on the grounds that they recorded a wide spread of melting temperatures of individual atoms measured by proton nuclear magnetic resonance (NMR) during their thermal denaturation. But their data are not of adequate quality to distinguish, within experimental error, between downhill folding and folding with a cooperative transition. Accordingly, their results offer no compelling evidence that Naf-BBL folds downhill, particularly as non-truncated, unmodified peripheral subunit binding domains seem to fold cooperatively