The relation between co- and post-translational protein folding and aggregation in the cell is poorly understood. Here, we employ a combination of fluorescence anisotropy decays in the frequency domain, fluorescence-detected solubility assays, and NMR spectroscopy to explore the role of the ribosome in protein folding within a biologically relevant context. First, we find that a primary function of the ribosome is to promote cotranslational nascent-protein solubility, thus supporting cotranslational folding even in the absence of molecular chaperones. Under these conditions, however, only a fraction of the soluble expressed protein is folded and freely tumbling in solution. Hence, the ribosome alone is insufficient to guarantee quantitative formation of the native state of the apomyoglobin (apoMb) model protein. Right after biosynthesis, nascent chains encoding apoMb emerge from the ribosomal exit tunnel and undergo a crucial irreversible post-translational kinetic partitioning between further folding and aggregation. Mutational analysis in combination with protein-expression kinetics and NMR show that nascent proteins can attain their native state only when the relative rates of soluble and insoluble product formation immediately upon release from the ribosome are tilted in favor of soluble species. Finally, the outcome of the above immediately post-translational kinetic partitioning is much more sensitive to amino acid sequence perturbations than the native fold, which is rather mutation-insensitive. Hence, kinetic channeling of nascent-protein conformation upon release from the ribosome may be a major determinant of evolutionary pressure.
The mechanism by which enzymes recognize the "uniform" collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Glyϳ[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P 3 subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.The degradation of collagen, the major structural component of connective tissues in skin, bone, tendon, and ligament, is an integral part in many biological processes such as wound healing, cell migration, tissue remodeling, and organ morphogenesis (1-4). Accelerated breakdown of collagen may result in many diseases such as arthritis, tumor cell invasion, glomerulonephritis, and metastasis (5-7). Types I, II, and III collagens, also called interstitial collagens, are the most abundant (8 -10), and contain a characteristic triple-helical conformation, which consists of three polyproline II-like helices supercoiled around a common axis (11,12). The close packing of the three chains can only accommodate Gly as every third residue, generating the repetitive (Gly-Xaa-Yaa) n sequence pattern. The Gly residues are all buried in the center, and the structure is stabilized by interchain N-H (Gly) . . . CϭO (Xaa) hydrogen bonds. The residues at the X and Y positions can be almost any amino acid, but they are frequently Pro and 4-hydroxyproline (Hyp 2 or O), respectively.The triple helical structure allows collagen to be degraded by only a few proteinases including a group of matrix metalloproteinases (MMPs) (5, 13). These MMPs (MMP-1, MMP-2, MMP-8, MMP-13, MMP-14, MMP-18) can bind and cleave interstitial collagens at a unique locus approximately threefourths away from the N terminus of the collagens (14). The cleavage site is after the Gly residue in the sequence of Glyϳ[Ile/Leu]-[Ala/Leu]...
Osteogenesis Imperfecta (OI), a hereditary connective tissue disease in collagen that arises from a single Gly->X mutation in the collagen chain, varies widely in phenotype from perinatal lethal to mild. It is unclear why there is such a large variation in the severity of the disease considering the repeating (Gly-X-Y)n sequence and the uniform rod-like structure of collagen. We systematically evaluate the effect of local (Gly-X-Y)n sequence around the mutation site on OI phenotype using integrated bio-statistical approaches, including odds ratio analysis and decision tree modeling. We show that different Gly->X mutations have different local sequence patterns that are correlated with lethal and nonlethal phenotypes providing a mechanism for understanding the sensitivity of local context in defining lethal and non-lethal OI. A number of important trends about which factors are related to OI phenotypes are revealed by the bio-statistical analyses; most striking is the complementary relationship between the placement of Pro residues and small residues and their correlation to OI phenotype. When Pro is present or small flexible residues are absent nearby a mutation site, the OI case tends to be lethal; when Pro is present or small flexible residues are absent further away from the mutation site, the OI case tends to be nonlethal. The analysis also reveals the dominant role of local sequence around mutation sites in the Major Ligand Binding Regions that are primarily responsible for collagen binding to its receptors and shows that non-lethal mutations are highly predicted by local sequence considerations alone whereas lethal mutations are not as easily predicted and may be a result of more complex interactions. Understanding the sequence determinants of OI mutations will enhance genetic counseling and help establish which steps in the collagen hierarchy to target for drug therapy.
Proteins are particularly prone to aggregation immediately after release from the ribosome, and it is therefore important to elucidate the role of chaperones during these key steps of protein life. The Hsp70 and trigger factor (TF) chaperone systems interact with nascent proteins during biogenesis and immediately post-translationally. It is unclear, however, whether these chaperones can prevent formation of soluble and insoluble aggregates. Here, we address this question by monitoring the solubility and structural accuracy of globin proteins biosynthesized in an Escherichia coli cell-free system containing different concentrations of the bacterial Hsp70 and TF chaperones. We find that Hsp70 concentrations required to grant solubility to newly synthesized proteins are extremely sensitive to client-protein sequence. Importantly, Hsp70 concentrations yielding soluble client proteins are insufficient to prevent formation of soluble aggregates. In fact, for some aggregation-prone protein variants, avoidance of soluble-aggregate formation demands Hsp70 concentrations that exceed cellular levels in E. coli. In all, our data highlight the prominent role of soluble aggregates upon nascentprotein release from the ribosome and show the limitations of the Hsp70 chaperone system in the case of highly aggregation-prone proteins. These results demonstrate the need to devise better strategies to prevent soluble-aggregate formation upon release from the ribosome.
Very little is known about the way proteins attain their native structure within the context of the living cell. In addition to the ribosome's well-established role in peptide bond formation, recent studies suggest that ribosomes play an important role in the early stages of protein folding in the cell and may be crucial for the production of folded bioactive proteins. Importantly, little is known about the impact of the mechanism of protein release from the ribosome on the attainment of a correctly folded conformation. Here, we present a kinetic study on the release time-course of fully synthesized ribosome-bound nascent proteins upon addition of the antibiotic puromycin. We focus these studies on the E. coli globin ApoHmpH. By time-resolved gel electrophoresis, we are able to follow puromycin's hydrolysis of the ester bond linking nascent polypeptides to the 3' end of tRNA. Steady-state fluorescence anisotropy allows us to follow the escape and folding of ApoHmpH from the ribosome. Finally, time decay fluorescence anisotropy analysis in the frequency domain complements the above techniques by providing insights into the local motions experienced by the nascent protein before and after release from the ribosome. Under experimental conditions where puromycin reacts at rates comparable to the naturally occurring release factors, we show that protein release from the ribosome is rate-limited by the C-terminal ester bond cleavage, and that escape from the ribosome and completion of folding occur quickly following this step. This result shows that the ribosomal context promotes a particularly ''temporally efficient'' folding upon nascent protein release. An important consequence of this phenomenon is the prevention of undesirable diffusion-and concentration-dependent phenomena such as aggregation. 579-Pos Board B359Diffusion Coefficient as a Function How well can the diffusion coefficient D of a globular biomolecule be predicted from its molecular mass MW? In ''Wanted: Scalable Tracers for Diffusion Measurements'' [J Phys Chem B, submitted], I propose that diffusion measurements in heterogeneous systems can be improved by the use of scalable tracers, in which the size is varied alone at constant shape, surface properties, diffusion mechanism, deformability, and other properties affecting diffusion. Before trying to design a de novo series of scalable globular proteins, it is appropriate to examine how scalable the commonly used de antiquo globular proteins are [ibid., supporting information]. The widely-used compilation of experimental diffusion coefficients by Tyn and Gusek [Biotech Bioeng 35 (1990) 327] was examined. This set -ranging from ribonuclease, 12640 Da, to tobacco mosaic virus, 50 MDa -was plotted as D versus log MW. The obviously linear species were removed, and values of D and MW for the outliers were examined. The plot yields a cloud of values of D versus log MW. In this plot, rigorously scalable tracers are expected to give a single smooth curve of D versus log MW, and the extent of the cloud represe...
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