Protein folding and assembly in a cell-free expression system

Department of Biochemistry, Texas A&M University, College Station 77843-2128, USA.
Methods in Enzymology (Impact Factor: 2.19). 01/1998; 290:1-17. DOI: 10.1016/S0076-6879(98)90003-9
Source: PubMed
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Very little is known about how protein structure evolves during the polypeptide chain elongation that accompanies cotranslational protein folding. This in vitro model study is aimed at probing how conformational space evolves for purified N-terminal polypeptides of increasing length. These peptides are derived from the sequence of an all-alpha-helical single domain protein, Sperm whale apomyoglobin (apoMb). Even at short chain lengths, ordered structure is found. The nature of this structure is strongly chain length dependent. At relatively short lengths, a predominantly non-native beta-sheet conformation is present, and self-associated amyloid-like species are generated. As chain length increases, alpha-helix progressively takes over, and it replaces the beta-strand. The observed trends correlate with the specific fraction of solvent-accessible nonpolar surface area present at different chain lengths. The C-terminal portion of the chain plays an important role by promoting a large and cooperative overall increase in helical content and by consolidating the monomeric association state of the full-length protein. Thus, a native-like energy landscape develops late during apoMb chain elongation. This effect may provide an important driving force for chain expulsion from the ribosome and promote nearly-posttranslational folding of single domain proteins in the cell. Nature has been able to overcome the above intrinsic misfolding trends by modulating the composition of the intracellular environment. An imbalance or improper functioning by the above modulating factors during translation may play a role in misfolding-driven intracellular disorders.
    Biochemistry 07/2003; 42(23):7090-9. DOI:10.1021/bi0273056 · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Aging is associated with extensive cognitive impairments, although the biochemical and physiological basis of these deficits are unknown. As the hippocampus plays a vital role in cognitive functions, we have selected this tissue to analyze changes in gene expression at two different ages. Array technology is utilized to explore how gene expression in hippocampus is affected by accelerated cognitive impairment in Senescence-Accelerated Mouse (SAM P8) strain. We show that the expression of genes associated with stress response and xenobiotic metabolism are strongly affected at a time when cognitive impairment occurs. Affected genes include those involved both in signaling and chaperone function. The effector and regulator family of chaperones, which play an important role in protein folding, and also the xenobiotic metabolizing enzymes that play crucial role in antioxidant systems, show significant changes in gene expression between 4 and 12 months.
    Biochemical and Biophysical Research Communications 07/2000; 272(3):657-61. DOI:10.1006/bbrc.2000.2719 · 2.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Biosynthetic folding, beginning with the growing nascent chain and leading to the biologically active structure within its proper cellular context, is one function shared by all proteins. We show that the bacterial luciferase beta subunit reaches its final native form in the alphabeta heterodimer much more rapidly during biosynthetic folding than during refolding from urea. The rate of formation of active enzyme is determined by a short-lived folding intermediate, which is able to associate with the alpha subunit very rapidly following release from the ribosome. This intermediate appears to involve a transient interaction of the C-terminal region of the beta subunit, a region distant from the subunit interface, but intimately involved in heterodimerization. Refolding of the beta subunit under similar conditions proceeds much more slowly. We have characterized both pathways and show that the basic difference between biosynthetic folding and refolding from urea is that the newly synthesized beta subunit enters the folding pathway at a point beyond the slow, rate-determining step that limits the rate of the renaturation process and constitutes a kinetic trap. This mechanism embodies a major strategy, the avoidance of slow-folding intermediates and kinetic traps, that may be employed by many proteins to achieve fast and efficient biosynthetic folding.
    Journal of Molecular Biology 11/1999; 294(2):579-86. DOI:10.1006/jmbi.1999.3281 · 3.96 Impact Factor