Sep 202011




M-PMV Protease








An electron density map








1) Structural biologists give unpublished structures to the CASP organizers.








Man over machine?




Mar 232010
ResearchBlogging.orgA protein has several different levels of structure. The primary structure is the arrangements of atoms and bonds, and it is formed in the ribosome by the assembly of amino acids as directed by an RNA template. The secondary structure is the local topology, the helices and strands, and this forms mostly because of the release of energy through the formation of hydrogen bonds. The tertiary structure is the actual fold of the protein, the way helices, strands, and loops are arranged in space. The fold forms primarily because of the favorable entropy of burying the protein’s hydrophobic groups where water cannot access them, analogous to the formation of an oil droplet in water. This suggests that, in addition to the well-known phenomenon of proteins denaturing, or losing their higher-order structure, under conditions of high heat, proteins might also denature when they get too cold.

As you might remember from your chemistry classes, the change in free energy due to a reaction under conditions of constant pressure is given by:
ΔG = ΔH – T ΔS
Where ΔH is the change in enthalpy (i.e. the heat released or absorbed by a reaction), ΔS is the change in entropy, and T is the temperature of the system in Kelvin. Here, the change we are talking about is the transition from the folded state to some unfolded state. Simplistically, since the entropic contribution is scaled by the temperature, one can imagine that for a reaction with favorable entropy and unfavorable enthalpy, lowering the temperature could cause the reaction to reverse. Protein folding is only marginally favorable at biological temperatures, so one could easily imagine that lowering the temperature enough could cause a protein to prefer the unfolded state.

Of course, this is an oversimplification: the entropy and enthalpy of a particular protein state do not remain constant over all temperatures. Rather, they vary in a way determined by the heat capacity (Cp), such that ΔG as a function of temperature is (1):

ΔG(T) = ΔH(Tr) + ΔCp(T-Tr) – T [ΔS(Tr) + ΔCp ln(T/Tr)]
Where Tr is some reference state at which the thermodynamic parameters have been determined, and ΔCp is defined with respect to the native (folded) state. Because the various states of the protein have different Cp (unfolded chains typically have higher Cp), at certain temperatures above and below the biological optimum we can expect proteins to lose their higher levels of structure. Even this is still an oversimplification, of course, because it does not directly account for changes in water structure and cosolute properties with temperature. These features may cause ΔCp itself to vary with temperature rather than remain constant.

Unfortunately, for most proteins the temperature that favors unfolding lies below the freezing point of water, which makes this phenomenon difficult to study unless you do something unusual to your system. In 2004, Babu et al. (1) reported results from experiments that used reverse micelles to study the denaturation of ubiquitin at temperatures below freezing. By encapsulating a protein-water droplet in inverted micelles dissolved in pentane, it was possible to reduce the temperature to 243 K without causing freezing. These micelles also had the convenient property of tumbling quickly in the pentane, which allowed for reasonable NMR spectra even at these low temperatures. The appearance of the spectra they obtained indicated that the protein underwent a slow unfolding process with many different unfolded states, and also that the protein did not unfold in a cooperative fashion. Rather, it appeared that one contiguous region of the protein unfolded while the rest remained folded (the main helix was particularly stable).

This wasn’t expected, because ubiquitin apparently unfolds in a completely two-state manner when overheated. This being the case, what’s expected is for the protein to either be all folded or all unfolded, not some mixture of the two. However, cold does not affect all intramolecular contacts the same way. Lowering the temperature is expected to make hydrophobic interactions less favorable while not significantly affecting polar interactions like hydrogen bonds. This being the case, one might expect an α-helix to persist through a cold-denaturation transition, as happens in this case.

Something similar is observed in an upcoming paper in JACS from the Raleigh and Eliezer Labs (2), which approaches cold denaturation using a mutant form of the C-terminal domain of ribosomal protein L9. An isoleucine to alanine mutation at residue 98 of this domain doesn’t appear to significantly alter the structure, but it causes the protein to denature somewhere in the high teens. At 12 °C the unfolded state is about 80% of the visible population, and this is where Shan et al. performed their NMR experiments. They assigned the unfolded state using standard techniques and then decided to see what they could learn from the chemical shifts.

As I’ve mentioned before, the chemical shift of a nucleus depends on the probability distribution of the surrounding electrons, and therefore is sensitive to the strength, composition, and angles of the atom’s chemical bonds. Because the dihedral angles of the protein backbone are a good proxy for the secondary structure, one can use the chemical shifts of particular atoms to determine whether a given residue is in a helix or strand. When they performed this analysis, Shan et al. noticed two major differences between the native and cold-denatured states of the protein. The first was that the helix and strand propensities of the denatured protein were much lower than the folded form, as expected. In addition, however, they noticed that one loop of the protein had gained α-helical character. That is, it seemed like an α-helix had actually gotten longer as a result of the unfolding.

This doesn’t mean that denaturing the protein added secondary structure. The low values in the output from the algorithm Shan et al. used suggest that the secondary structure in this denatured state forms only transiently. However, the chemical shifts suggest, and other structural data appear to confirm, that this region of the protein has an increased propensity to inhabit a helical structure as a consequence of the unfolding.

These results emphasize the fact that the “unfolded state” isn’t as simple as it’s often described. Residual structure persists in unfolded states of many proteins, and unfolded ensembles of one protein generated through different means (heat, cold, pH, cosolutes) may not resemble each other. Unlike unfolding at high temperature, cold denaturation of ubiquitin appears to be non-cooperative. In both ubiquitin and L9, it appears that helices are robust to the unfolding process, persisting and even propagating as the protein denatures. While some of these features may be held in common between different kinds of denatured states, others may be unique to particular denaturation conditions. The lingering question is which of these unfolded ensembles best resembles the denatured state that exists under biological conditions, giving rise to misfolded states and their associated diseases.

(1) Babu, C., Hilser, V., & Wand, A. (2004). Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation Nature Structural & Molecular Biology, 11 (4), 352-357 DOI: 10.1038/nsmb739

(2) Shan, B., McClendon, S., Rospigliosi, C., Eliezer, D., & Raleigh, D. (2010). The Cold Denatured State of the C-terminal Domain of Protein L9 Is Compact and Contains Both Native and Non-native Structure Journal of the American Chemical Society DOI: 10.1021/ja908104s

 Posted by at 10:00 PM
Aug 182009





0,0 0,y 0,2y 0,3y … 0,my



and so on until








Mar 242009
ResearchBlogging.orgThe ribosome produces proteins by matching tRNA that has been correctly loaded with an amino acid to a codon (triplet of DNA bases) in the mRNA that contains the gene sequence. The triplet code allows 64 combinations of nucleotide bases, but proteins are made from only 20 amino acids (plus a “stop” signal). This means that most amino acids are coded by multiple codons, and hence have multiple tRNAs. Not all codons are created equal, however; in bacteria some codons are found much less frequently than others that represent the same amino acid. The tRNA associated with these “rare codons” is also less abundant than other tRNA, and this means that when a ribosome hits a rare codon, it often has to pause while it waits to encounter a loaded tRNA. To structural biologists like myself, who do their work by overexpressing proteins in bacteria, rare codons can be a nuisance because they slow down protein production, or even prevent it entirely. In a recent paper in Nature Structural & Molecular Biology, however, researchers from Germany suggest that the slowdown due to rare codons may have a functional advantage in vivo.

As a first step, Zhang et al. used a bioinformatics approach to survey the sequences of bacterial genes so that they could identify patches that would be slow to translate (the Methods section appears to contain an error in the description of this technique). They found that for proteins longer than about 300 amino acid residues, nearly every transcript contained at least one cluster of slow-translating codons. When the authors used a cell-free E. coli expression system to make some of these proteins and allowed only one round of translation initiation per ribosome, they saw a pattern of translation intermediates that matched the sizes predicted by the location of slow-translating patches.

In order to find out whether these translation intermediates had any significance, the authors examined the multi-domain protein SufI. In their prediction of the translation speed, which is on top in this figure that I have shamelessly stolen, there are four slow spots. Aside from the first one, these appear to correspond to the boundaries of different structural domains in the protein (lower part of the figure). Experiments with proteases suggested that these domains actually folded during the pauses, as the ribosome-bound translation intermediates were resistant to proteolysis.

Interestingly, when two rare leucine codons were replaced by more common ones (the authors call this SufIΔ25-28), the whole protein became vulnerable to degradation. Similarly, when extra tRNA for these rare codons was added to the cell-free expression system, the full-length protein became protease-sensitive. This suggests that the slow patches are actually necessary for proper folding of the protein. It’s often the case that lowering the incubation temperature can improve the expression of certain proteins in E. coli. The authors of this study find that is also true for SufI, as the protease resistance of SufIΔ25-28 can be restored by lowering the temperature, and thus the overall translation rate. When analogous experiments with SufIΔ25-28 and tRNA supplementation were carried out in living E. coli, the translocation of SufI into the periplasmic space was reduced by a factor of 10 even though the overall protein concentration was not affected, indicating that the co-translational folding allowed by the rare codons is necessary for proper functioning of the protein in vivo.

Of course this is a single case study, and it would be premature to conclude that every patch of rare codons corresponds to an important co-translational folding event. Indeed, that doesn’t even appear to be true of SufI, which folds properly when one of its other slow patches is removed. However, at certain key locations these stretches of rare codons may be an important part of the folding machinery in multidomain proteins. In addition, the more frequent appearance of rare codons in β-strands (as opposed to α-helices) may also be related to folding due to the slower kinetics of β-sheet formation. As the authors note, the intrinsic kinetics aren’t everything — pauses in the translation process may also buy time for the complex to encounter essential chaperones or cofactors. Regardless of the mechanism, it appears that rare codons, in at least some instances, provide a way for the folding process to catch up with the translation process.

Zhang, G., Hubalewska, M., & Ignatova, Z. (2009). Transient ribosomal attenuation coordinates protein synthesis and co-translational folding Nature Structural & Molecular Biology, 16 (3), 274-280 DOI: 10.1038/nsmb.1554

 Posted by at 1:00 AM