Biomedical subjects

George Stan

Publications and source records attributed to George Stan.

4 recordsLinked to original sources

PubMed2006

Residues in substrate proteins that interact with GroEL in the capture process are buried in the native state.

We have used a bioinformatic approach to predict the natural substrate proteins for the Escherichia coli chaperonin GroEL based on two simple criteria. Natural substrate proteins should contain binding motifs similar in sequence to the mobile loop peptide of GroES that displaces the binding motif during the chaperonin cycle. Secondly, each substrate protein should contain multiple copies of the binding motif so that the chaperonin can perform "work" on the substrate protein. To validate these criteria, we have used a database of 252 proteins that have been experimentally shown to interact with the chaperonin machinery in vivo. More than 80% are identified by these criteria. The binding motifs of all 79 proteins in the database with a known three-dimensional structure are buried (<50% solvent-accessible surface area) in the native state. Our results show that the binding motifs are inaccessible in the native state but become solvent-exposed in unfolded state, thus enabling GroEL to distinguish between unfolded and native states. The structures of the binding motif in the native states of the substrate proteins include alpha-helices, beta-strands, and random coils. The diversity of secondary structures implies that there are large and varied conformational transitions in the recognition motifs after their displacement by the mobile loops of GroES.

Binding Sites↗

PubMed2005

Probing the "annealing" mechanism of GroEL minichaperone using molecular dynamics simulations.

Although the intact chaperonin machinery is needed to rescue natural substrate proteins (SPs) under non-permissive conditions the "minichaperone" alone, containing only the isolated apical domain of GroEL, can assist folding of a certain class of proteins. To understand the annealing function of the minichaperone, we have carried out molecular dynamics simulations in the NPT ensemble totaling 300ns for four systems; namely, the isolated strongly binding peptide (SBP), the minichaperone, and the SBP and a weakly binding peptide (WBP) in complex with the minichaperone. The SBP, which is structureless in isolation, adopts a beta-hairpin conformation in complex with the minichaperone suggesting that favorable non-specific interactions of the SPs confined to helices H and I of the apical domains can induce local secondary structures. Comparison of the dynamical fluctuations of the apo and the liganded forms of the minichaperone shows that the stability (needed for SP capture) involves favorable hydrophobic interactions and hydrogen bond network formation between the SBP and WBP, and helices H and I. The release of the SP, which is required for the annealing action, involves water-mediated interactions of the charged residues at the ends of H and I helices. The simulation results are consistent with a transient binding release (TBR) model for the annealing action of the minichaperone. According to the TBR model, SP annealing occurs in two stages. In the first stage the SP is captured by the apical domain. This is followed by SP release (by thermal fluctuations) that places it in a different region of the energy landscape from which it can partition rapidly to the native state with probability Phi or be trapped in another misfolded state. The process of binding and release can result in enhancement of the native state yield. The TBR model suggests "that any cofactor that can repeatedly bind and release SPs can be effective in assisting protein folding." By comparing the structures of the non-chaperone alpha-casein (which has no sequence similarity with the apical domain) and the minichaperone and the hydrophobicity profiles we show that alpha-casein has a pair of helices that have similar sequence and structural profiles as H and I. Based on this comparison we identify residues that stabilize (destabilize) alpha-casein-protein complexes. This suggests that alpha-casein assists folding by the TBR mechanism.

Animals↗

PubMed2004

Identifying natural substrates for chaperonins using a sequence-based approach.

The Escherichia coli chaperonin machinery, GroEL, assists the folding of a number of proteins. We describe a sequence-based approach to identify the natural substrate proteins (SPs) for GroEL. Our method is based on the hypothesis that natural SPs are those that contain patterns of residues similar to those found in either GroES mobile loop and/or strongly binding peptide in complex with GroEL. The method is validated by comparing the predicted results with experimentally determined natural SPs for GroEL. We have searched for such patterns in five genomes. In the E. coli genome, we identify 1422 (about one-third) sequences that are putative natural SPs. In Saccharomyces cerevisiae, 2885 (32%) of sequences can be natural substrates for Hsp60, which is the analog of GroEL. The precise number of natural SPs is shown to be a function of the number of contacts an SP makes with the apical domain (N(C)) and the number of binding sites (N(B)) in the oligomer with which it interacts. For known SPs for GroEL, we find approximately 4 < N(C) < 5 and 2 <or= N(B) <or= 4. A limited analysis of the predicted binding sequences shows that they do not adopt any preferred secondary structure. Our method also predicts the putative binding regions in the identified SPs. The results of our study show that a variety of SPs, associated with diverse functions, can interact with GroEL.

Binding Sites↗

PubMed2003

Annealing function of GroEL: structural and bioinformatic analysis.

The Escherichia coli chaperonin system, GroEL-GroES, facilitates folding of substrate proteins (SPs) that are otherwise destined to aggregate. The iterative annealing mechanism suggests that the allostery-driven GroEL transitions leading to changes in the microenvironment of the SP constitutes the annealing action of chaperonins. To describe the molecular basis for the changes in the nature of SP-GroEL interactions we use the crystal structures of GroEL (T state), GroEL-ATP (R state) and the GroEL-GroES-(ADP)(7) (R" state) complex to determine the residue-specific changes in the accessible surface area and the number of tertiary contacts as a result of the T-->R-->R" transitions. We find large changes in the accessible area in many residues in the apical domain, but relatively smaller changes are associated with residues in the equatorial domain. In the course of the T-->R transition the microenvironment of the SP changes which suggests that GroEL is an annealing machine even without GroES. This is reflected in the exposure of Glu386 which loses six contacts in the T-->R transition. We also evaluate the conservation of residues that participate in the various chaperonin functions. Multiple sequence alignments and chemical sequence entropy calculations reveal that, to a large extent, only the chemical identities and not the residues themselves important for the nominal functions (peptide binding, nucleotide binding, GroES and substrate protein release) are strongly conserved. Using chemical sequence entropy, which is computed by classifying aminoacids into four types (hydrophobic, polar, positively charged and negatively charged) we make several new predictions that are relevant for peptide binding and annealing function of GroEL. We identify a number of conserved peptide binding sites in the apical domain which coincide with those found in the 1.7 A crystal structure of 'mini-chaperone' complexed with the N-terminal tag. Correlated mutations in the HSP60 family, that might control allostery in GroEL, are also strongly conserved. Most importantly, we find that charged solvent-exposed residues in the T state (Lys 226, Glu 252 and Asp 253) are strongly conserved. This leads to the prediction that mutating these residues, that control the annealing function of the SP, can decrease the efficacy of the chaperonin function.

Algorithms↗