Biomedical subjects

Hung-Chun Chang

Publications and source records attributed to Hung-Chun Chang.

6 recordsLinked to original sources

PubMed2006

Real-time observation of trigger factor function on translating ribosomes.

The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of approximately 10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.

Escherichia coli Proteins↗

PubMed2006

Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein.

GroEL and GroES form a chaperonin nano-cage for proteins up to approximately 60 kDa to fold in isolation. Here we explored the structural features of the chaperonin cage critical for rapid folding of encapsulated substrates. Modulating the volume of the GroEL central cavity affected folding speed in accordance with confinement theory. Small proteins (approximately 30 kDa) folded more rapidly as the size of the cage was gradually reduced to a point where restriction in space slowed folding dramatically. For larger proteins (approximately 40-50 kDa), either expanding or reducing cage volume decelerated folding. Additionally, interactions with the C-terminal, mildly hydrophobic Gly-Gly-Met repeat sequences of GroEL protruding into the cavity, and repulsion effects from the negatively charged cavity wall were required for rapid folding of some proteins. We suggest that by combining these features, the chaperonin cage provides a physical environment optimized to catalyze the structural annealing of proteins with kinetically complex folding pathways.

Amino Acid Sequence↗

PubMed2005

De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria.

Eukaryotic genomes encode a considerably higher fraction of multi-domain proteins than their prokaryotic counterparts. It has been postulated that efficient co-translational and sequential domain folding has facilitated the explosive evolution of multi-domain proteins in eukaryotes by the recombination of pre-existent domains. Here, we tested whether eukaryotes and bacteria differ generally in the folding efficiency of multi-domain proteins generated by domain recombination. To this end, we compared the folding behavior of a series of recombinant proteins comprised of green fluorescent protein (GFP) fused to four different robustly folding proteins through six different linkers upon expression in Escherichia coli and the yeast Saccharomyces cerevisiae. We found that, unlike yeast, bacteria are remarkably inefficient at folding these fusion proteins, even at comparable levels of expression. In vitro and in vivo folding experiments demonstrate that the GFP domain imposes significant constraints on de novo folding of its fusion partners in bacteria, consistent with a largely post-translational folding mechanism. This behavior may result from an interference of GFP with adjacent domains during folding due to the particular topology of the beta-barrel GFP structure. By following the accumulation of enzymatic activity, we found that the rate of appearance of correctly folded fusion protein per ribosome is indeed considerably higher in yeast than in bacteria.

Escherichia coli↗

PubMed2005

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli.

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.

Chaperonin 10↗

PubMed2005

Regulation of Hsp70 function by HspBP1: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange.

HspBP1 belongs to a family of eukaryotic proteins recently identified as nucleotide exchange factors for Hsp70. We show that the S. cerevisiae ortholog of HspBP1, Fes1p, is required for efficient protein folding in the cytosol at 37 degrees C. The crystal structure of HspBP1, alone and complexed with part of the Hsp70 ATPase domain, reveals a mechanism for its function distinct from that of BAG-1 or GrpE, previously characterized nucleotide exchange factors of Hsp70. HspBP1 has a curved, all alpha-helical fold containing four armadillo-like repeats unlike the other nucleotide exchange factors. The concave face of HspBP1 embraces lobe II of the ATPase domain, and a steric conflict displaces lobe I, reducing the affinity for nucleotide. In contrast, BAG-1 and GrpE trigger a conserved conformational change in lobe II of the ATPase domain. Thus, nucleotide exchange on eukaryotic Hsp70 occurs through two distinct mechanisms.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed.

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

Escherichia coli↗