Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel.

In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1-140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63-94 residues. Longer RNCs (apoHmp1-189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.

Selective Isotope Labeling and LC-Photo-CIDNP Enable NMR Spectroscopy at Low-Nanomolar Concentration.

NMR spectroscopy is a powerful tool to investigate molecular structure and dynamics. The poor sensitivity of this technique, however, limits its ability to tackle questions requiring dilute samples. Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an optically enhanced NMR technology capable of addressing the above challenge by increasing the detection limit of aromatic amino acids in solution up to 1000-fold, either in isolation or within proteins. Here, we show that the absence of NMR-active nuclei close to a magnetically active site of interest (e.g., the structurally diagnostic 1Hα-13Cα pair of amino acids) is expected to significantly increase LC-photo-CIDNP hyperpolarization. Then, we exploit the spin-diluted tryptophan isotopolog Trp-α-13C-ß,ß,2,4,5,6,7-d7 and take advantage of the above prediction to experimentally achieve a ca 4-fold enhancement in NMR sensitivity over regular LC-photo-CIDNP. This advance enables the rapid (within seconds) detection of 20 nM concentrations or the molecule of interest, corresponding to a remarkable 3 ng detection limit. Finally, the above Trp isotopolog is amenable to incorporation within proteins and is readily detectable at a 1 µM concentration in complex cell-like media, including Escherichia coli cell-free extracts.

An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel.

The influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences.

Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis.

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.

Stabilization of the Reductase Domain in the Catalytically Self-Sufficient Cytochrome P450BM3 by Consensus-Guided Mutagenesis.

The multidomain, catalytically self-sufficient cytochrome P450 BM-3 from Bacillus megaterium (P450BM3 ) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus-guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450BM3 . Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild-type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half-lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD-binding domain and that the optimal temperature (Topt ) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their ß-turn occurrence, we engineered two chimerical enzymes where their super secondary ß-loop-α motifs 2 ((ßα)2 ) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (ßα)2 motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (ßα)2 motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (ßα)2 of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N-terminal of loop-2 and the C-terminal of α-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (EA ). A systematic analysis of DSC data showed a large decrease on the EA of TcTIM (ΔEA ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571-579. © 2016 Wiley Periodicals, Inc.

Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

BACKGROUND: The glycolytic pathway plays an important role in tumor cells. Triosephosphate isomerase (TIM) catalyzes the reversible isomerization of D-glyceraldehyde-3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) in the glycolysis. Proteomics of a human prostate adenocarcinoma cell line revealed the presence of the G233D TIM variant, a new allelic type whose biochemical properties have not been reported [1]. OBJECTIVE: Provide the first biochemical and biophysical characterization of the allelic variant G233D of TIM. METHODS: The Michaelis-Menten curves using both substrates of TIM were obtained. Also the effect of the competitive inhibitor phosphoenolpyruvate (PEP) was assessed in presence of GAP and DHAP. The thermal stability in absence and presence of PEP was analyzed by circular dichroism spectroscopy. For comparison purposes, all the measurements were carried out on the wild type TIM and variant G233D. RESULTS: The G233D variant exhibited a kcat value 4-fold lower than that of the WT enzyme in the GAP isomerization to DHAP, which is the reverse reaction of the glycolytic pathway. The G233D variant exhibited Ki and IC50 values of 120 µM and 356 µM in the presence of several concentrations of GAP and 0.3 mM DHAP, respectively. These inhibition parameters are similar to those exhibited by the WT enzyme. The thermal unfolding cooperativity of G233D variant was significantly increased upon PEP binding, suggesting that the ligand-bound enzyme was trapped in a rigid conformation. CONCLUSION: We suggest that the flow of GAP through glycolysis could be enhanced by the decreased activity of the G233D variant in the formation of DHAP.

The folding pathway of glycosomal triosephosphate isomerase: structural insights into equilibrium intermediates.

The guanidine hydrochloride-induced conformational transitions of glycosomal triosephosphate isomerase (TIM) were monitored with functional, spectroscopic, and hydrodynamic measurements. The equilibrium folding pathway was found to include two intermediates (N(2) ↔I(2) ↔2M↔2U). According to this model, the conformational stability parameters of TIM are as follows: ΔG(I2-N2) = 5.5 ± 0.6, ΔG(2M-I2) =19.6 ± 1.6, and ΔG(2U-2M) = 14.7 ± 3.1 kcal mol(-1) . The I(2) state is compact (α(SR) = 0.8); it is able to bind 8-anilinonaphthalene-1-sulfonic acid ANS and it is composed of ∼45% of α-helix and tertiary structure content compared with the native enzyme; however, it is unable to bind the transition-state analog 2-phosphoglycolate. Conversely, the 2M state lacks detectable tertiary contacts, possesses ∼10% of the native α-helical content, is significantly expanded (α(SR) = 0.2), and has low affinity for ANS. We studied the effect of mutating cysteine residues on the structure and stability of I(2) and 2M. Three mutants were made: C39A, C126A, and C39A/C126A. The replacement of C39, which is located at ß(2) , was found to be neutral. The I(2) -C126A state, however, was prone to aggregation and exhibited an emission maximum that was 3-nm red-shifted compared with the I(2) -wild type, indicating solvent exposure of W90 at ß(4) . Our results suggest that the I(2) state comprises the (ßα)(1-4) ß(5) module in which the conserved C126 residue located at ß(5) defines the boundary of the folded segment. We propose a folding pathway that highlights the remarkable thermodynamic stability of this glycosomal enzyme.