A natively unfolded toxin domain uses its receptor as a folding template

Natively unfolded proteins range from molten globules to disordered coils. They are abundant in eukaryotic genomes and commonly involved in molecular interactions. The essential N-terminal translocation domains of colicin toxins from Escherichia coli are disordered bacterial proteins that bind at least one protein of the Tol or Ton family. The colicin N translocation domain (ColN-(1-90)), which binds to the C-terminal domain of TolA (TolA-(296-421)), shows a disordered far-UV CD spectrum, no near-UV CD signal, and non-cooperative thermal unfolding. As expected, TolA-(296-421) displays both secondary structure in far-UV CD and tertiary structure in near-UV CD. Furthermore it shows a cooperative unfolding transition at 65 degrees C. CD spectra of the 1:1 complex show both increased secondary structure and colicin N-specific near-UV CD signals. A new cooperative thermal transition at 35 degrees C is followed by the unchanged unfolding behavior of TolA-(296-421). Fluorescence and surface plasmon resonance confirm that the new unfolding transition accompanies dissociation of ColN-(1-90). Hence upon binding the disordered structure of ColN-(1-90) converts to a cooperatively folded domain without altering the TolA-(296-421) structure.     

Characterizing the unstructured intermediates in oxidative folding

A recently developed method is used here to characterize some of the folding intermediates, and the oxidative folding processes, of RNase A. This method is based on the ability of trans-[Pt(en)(2)Cl(2)](2+) to oxidize cysteine residues to form disulfide bonds faster than the disulfide bonds can be rearranged by reshuffling or reduction. Variations of this method have enabled us to address three issues. (i) How the nature of the residual structure and/or conformational order that is present, or develops, during the initial stages of folding can be elucidated. It is shown here that there is a 10-fold increase in the propensity of the unfolded reduced forms of RNase A to form the native set of disulfides directly, compared to the propensity under strongly denaturing conditions (4-6 M GdnHCl). Thus, the unfolded reduced forms of RNase A are not statistical coils with a more condensed form than in the GdnHCl-denatured state; rather, it is suggested that reduced RNase A has a little bias toward a native topology. (ii) The structural characterization of oxidative folding intermediates in terms of disulfide pairing is demonstrated; specifically, a lower-limit estimate is made of the percentage of native disulfide-containing molecules in the two-disulfide ensemble of RNase A. (iii) The critical role of structured intermediate species in determining the oxidative folding pathways of proteins was shown previously. Here, we demonstrate that the presence of a structured intermediate in the oxidative folding of proteins can be revealed by this method.     

Coupling of folding and binding for unstructured proteins

There are now numerous examples of proteins that are unstructured or only partially structured under physiological conditions and yet are nevertheless functional. Such proteins are especially prevalent in eukaryotes. In many cases, intrinsically disordered proteins adopt folded structures upon binding to their biological targets. Many new examples of coupled folding and binding events have been reported recently, providing new insights into mechanisms of molecular recognition.     

Consequences of stabilizing the natively disordered f helix for the folding pathway of apomyoglobin

The F helix region of sperm whale apomyoglobin is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. To examine the effects of F helix stabilization on the folding pathway of apomyoglobin, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared: a double mutant, P88K/S92K (F2), and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst-phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.     

Requirement for natively unstructured regions of mesoderm development candidate 2 in promoting low-density lipoprotein receptor-related protein 6 maturation

Proteins of the low-density lipoprotein receptor family (LRPs) are complex, multimodular type I transmembrane receptors. Productive maturation of these proteins relies on an ER-resident protein called mesoderm development candidate 2 (MESD) in mammals and Boca in Drosophila. We show here that MESD contains a central folded domain flanked by natively unstructured regions required to facilitate maturation of LRP6. Enforced expression of full-length human MESD promotes the secretion of soluble minireceptors derived from LRP6 that contain either one or two beta-propeller-EGF domain pairs. Conversely, siRNA-mediated knockdown of human MESD expression blocks secretion of native LRP6 minireceptors and dramatically reduces the level of cell-surface expression of full-length LRP6. Cell-surface expression is only rescued by simultaneous delivery of siRNA-resistant forms of mouse MESD that contain most or all of the unstructured N- and C-termini, implicating the flexible parts of MESD in its function of promoting LRP maturation.     

Fesselin is a natively unfolded protein

Fesselin is a heat stable proline-rich actin binding protein. The stability, amino acid composition, and ability to bind to several proteins suggested that fesselin may be unfolded under native conditions. While the complete sequence of fesselin is unknown an analysis of a closely related protein, synaptopodin 2 from Gallus gallus, indicates that fesselin consists of a series of unstructured regions interspersed between short folded regions. To determine if fesselin is natively unfolded, we compared fesselin to a known globular protein (myosin S1) and a known unfolded protein Cad22 (the COOH terminal 22 kDa fragment of caldesmon). Fesselin, and Cad22, had larger Stokes radii than globular proteins of equivalent mass. The environments of tryptophan residues of fesselin and Cad22 were the same in the presence and absence of 6 M guanidine hydrochloride. Fesselin had a circular dichroism spectrum that was primarily random coil. Changes in pH over the range of 1.5-11.5 did not alter that spectrum. Increasing the temperature to 85 degrees C caused an increase in the degree of secondary structure. Calmodulin binding to fesselin altered the environment of the tryptophan residues so that they became less sensitive to the quencher acrylamide. These results show that fesselin is a natively unfolded protein.