Tissue- and development-stage-specific mRNA and heterogeneous CNV signatures of human ribosomal proteins in normal and cancer samples.

We give results from a detailed analysis of human Ribosomal Protein (RP) levels in normal and cancer samples and cell lines from large mRNA, copy number variation and ribosome profiling datasets. After normalizing total RP mRNA levels per sample, we find highly consistent tissue specific RP mRNA signatures in normal and tumor samples. Multiple RP mRNA-subtypes exist in several cancers, with significant survival and genomic differences. Some RP mRNA variations among subtypes correlate with copy number loss of RP genes. In kidney cancer, RP subtypes map to molecular subtypes related to cell-of-origin. Pan-cancer analysis of TCGA data showed widespread single/double copy loss of RP genes, without significantly affecting survival. In several cancer cell lines, CRISPR-Cas9 knockout of RP genes did not affect cell viability. Matched RP ribosome profiling and mRNA data in humans and rodents stratified by tissue and development stage and were strongly correlated, showing that RP translation rates were proportional to mRNA levels. In a small dataset of human adult and fetal tissues, RP protein levels showed development stage and tissue specific heterogeneity of RP levels. Our results suggest that heterogeneous RP levels play a significant functional role in cellular physiology, in both normal and disease states.

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and they require chaperones to keep them soluble and translocation competent. Here we show that a novel targeting factor in the chloroplast signal recognition particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to 'ATPases associated with various cellular activities' (AAA(+)) chaperones, cpSRP43 uses specific binding interactions with its substrate to mediate its 'disaggregase' activity. This disaggregase capability can allow targeting machineries to more effectively capture their protein substrates and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example to our knowledge of an ATP-independent disaggregase and shows that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate.

Dynamic charge interactions create surprising rigidity in the ER/K alpha-helical protein motif.

Protein alpha-helices are ubiquitous secondary structural elements, seldom considered to be stable without tertiary contacts. However, amino acid sequences in proteins that are based on alternating repeats of four glutamic acid (E) residues and four positively charged residues, a combination of arginine (R) and lysine (K), have been shown to form stable alpha-helices in a few proteins, in the absence of tertiary interactions. Here, we find that this ER/K motif is more prevalent than previously reported, being represented in proteins of diverse function from archaea to humans. By using molecular dynamics (MD) simulations, we characterize a dynamic pattern of side-chain interactions that extends along the backbone of ER/K alpha-helices. A simplified model predicts that side-chain interactions alone contribute substantial bending rigidity (0.5 pN/nm) to ER/K alpha-helices. Results of small-angle x-ray scattering (SAXS) and single-molecule optical-trap analyses are consistent with the high bending rigidity predicted by our model. Thus, the ER/K alpha-helix is an isolated secondary structural element that can efficiently span long distances in proteins, making it a promising tool in designing synthetic proteins. We propose that the significant rigidity of the ER/K alpha-helix can help regulate protein function, as a force transducer between protein subdomains.

Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model.

Hepatitis C virus NS3 helicase is an enzyme that unwinds double-stranded polynucleotides in an ATP-dependent reaction. It provides a promising target for small molecule therapeutic agents against hepatitis C. Design of such drugs requires a thorough understanding of the dynamical nature of the mechanochemical functioning of the helicase. Despite recent progress, the detailed mechanism of the coupling between ATPase activity and helicase activity remains unclear. Based on an elastic network model (ENM), we apply two computational analysis tools to probe the dynamical mechanism underlying the allosteric coupling between ATP binding and polynucleotide binding in this enzyme. The correlation analysis identifies a network of hot-spot residues that dynamically couple the ATP-binding site and the polynucleotide-binding site. Several of these key residues have been found by mutational experiments as functionally important, while our analysis also reveals previously unexplored hot-spot residues that are potential targets for future mutational studies. The conformational changes between different crystal structures of NS3 helicase are found to be dominated by the lowest frequency mode solved from the ENM. This mode corresponds to a hinge motion of the highly flexible domain 2. This motion simultaneously modulates the opening/closing of the domains 1-2 cleft where ATP binds, and the domains 2-3 cleft where the polynucleotide binds. Additionally, a small twisting motion of domain 1, observed in both mode 1 and the computed ATP binding induced conformational change, fine-tunes the binding affinity of the domains 1-3 interface for the polynucleotide. The combination of these motions facilitates the translocation of a single-stranded polynucleotide in an inchworm-like manner.

How large is an alpha-helix? Studies of the radii of gyration of helical peptides by small-angle X-ray scattering and molecular dynamics.

Using synchrotron radiation and the small-angle X-ray scattering technique we have measured the radii of gyration of a series of alanine-based alpha-helix-forming peptides of the composition Ace-(AAKAA)(n)-GY-NH(2), n=2-7, in aqueous solvent at 10(+/-1) degrees C. In contrast to other techniques typically used to study alpha-helices in isolation (such as nuclear magnetic resonance and circular dichroism), small-angle X-ray scattering reports on the global structure of a molecule and, as such, provides complementary information to these other, more sequence-local measuring techniques. The radii of gyration that we measure are, except for the 12-mer, lower than the radii of gyration of ideal alpha-helices or helices with frayed ends of the equivalent sequence-length. For example, the measured radius of gyration of the 37-mer is 14.2(+/-0.6)A, which is to be compared with the radius of gyration of an ideal 37-mer alpha-helix of 17.6A. Attempts are made to analyze the origin of this discrepancy in terms of the analytical Zimm-Bragg-Nagai (ZBN) theory, as well as distributed computing explicit solvent molecular dynamics simulations using two variants of the AMBER force-field. The ZBN theory, which treats helices as cylinders connected by random walk segments, predicts markedly larger radii of gyration than those measured. This is true even when the persistence length of the random walk parts is taken to be extremely short (about one residue). Similarly, the molecular dynamics simulations, at the level of sampling available to us, give inaccurate values of the radii of gyration of the molecules (by overestimating them by around 25% for longer peptides) and/or their helical content. We conclude that even at the short sequences examined here (< or =37 amino acid residues), these alpha-helical peptides behave as fluctuating semi-broken rods rather than straight cylinders with frayed ends.

Unusual compactness of a polyproline type II structure.

Polyproline type II (PPII) helix has emerged recently as the dominant paradigm for describing the conformation of unfolded polypeptides. However, most experimental observables used to characterize unfolded proteins typically provide only short-range, sequence-local structural information that is both time- and ensemble-averaged, giving limited detail about the long-range structure of the chain. Here, we report a study of a long-range property: the radius of gyration of an alanine-based peptide, Ace-(diaminobutyric acid)2-(Ala)7-(ornithine)2-NH2. This molecule has previously been studied as a model for the unfolded state of proteins under folding conditions and is believed to adopt a PPII fold based on short-range techniques such as NMR and CD. By using synchrotron radiation and small-angle x-ray scattering, we have determined the radius of gyration of this peptide to be 7.4 +/- 0.5 angstroms, which is significantly less than the value expected from an ideal PPII helix in solution (13.1 angstroms). To further study this contradiction, we have used molecular dynamics simulations using six variants of the AMBER force field and the GROMOS 53A6 force field. However, in all cases, the simulated ensembles underestimate the PPII content while overestimating the experimental radius of gyration. The conformational model that we propose, based on our small angle x-ray scattering results and what is known about this molecule from before, is that of a very flexible, fluctuating structure that on the level of individual residues explores a wide basin around the ideal PPII geometry but is never, or only rarely, in the ideal extended PPII helical conformation.

Random-coil behavior and the dimensions of chemically unfolded proteins.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R(G)) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R(G) of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r(2) = 0.988) by a power-law relationship with a best-fit exponent, 0.598 +/- 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

A comparative study of motor-protein motions by using a simple elastic-network model.

In this work, we report on a study of the structure-function relationships for three families of motor proteins, including kinesins, myosins, and F1-ATPases, by using a version of the simple elastic-network model of large-scale protein motions originally proposed by Tirion [Tirion, M. (1996) Phys. Rev. Lett. 77, 1905-1908]. We find a surprising dichotomy between kinesins and the other motor proteins (myosins and F1-ATPase). For the latter, there exist one or two dominant lowest-frequency modes (one for myosin, two for F1-ATPase) obtained from normal-mode analysis of the elastic-network model, which overlap remarkably well with the measured conformational changes derived from pairs of solved crystal structures in different states. Furthermore, we find that the computed global conformational changes induced by the measured deformation of the nucleotide-binding pocket also overlap well with the measured conformational changes, which is consistent with the "nucleotide-binding-induced power-stroke" scenario. In contrast, for kinesins, this simplicity breaks down. Multiple modes are needed to generate the measured conformational changes, and the computed displacements induced by deforming the nucleotide-binding pocket also overlap poorly with the measured conformational changes, and are insufficient to explain the large-scale motion of the relay helix and the linker region. This finding may suggest the presence of two different mechanisms for myosins and kinesins, despite their strong evolutionary ties and structural similarities.

Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis.

Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.

Equilibrium collapse and the kinetic 'foldability' of proteins.

An important element of protein folding theory has been the identification of equilibrium parameters that might uniquely distinguish rapidly folding polypeptide sequences from those that fold slowly. One such parameter, termed sigma, is a dimensionless, equilibrium measure of the coincidence of chain compaction and folding that is predicted to be an important determinant of relative folding kinetics. To test this prediction and improve our understanding of the putative relationship between nonspecific compaction of the unfolded state and protein folding kinetics, we have used small-angle X-ray scattering and circular dichroism spectroscopy to measure the sigma of five well-characterized proteins. Consistent with theoretical predictions, we find that near-perfect coincidence of the unfolded state contraction and folding (sigma approximately 0) is associated with the rapid kinetics of these naturally occurring proteins. We do not, however, observe any significant correlation between sigma and either the relative folding rates of these proteins or the presence or absence of well-populated kinetic intermediates. Thus, while sigma approximately 0 may be a necessary condition to ensure rapid folding, differences in sigma do not account for the wide range of rates and mechanisms with which naturally occurring proteins fold.

Elucidation of the molecular mechanism during the early events in immunoglobulin light chain amyloid fibrillation. Evidence for an off-pathway oligomer at acidic pH.

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.

Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH.

Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivo fibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability, and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 m urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.