Fluorotryptophan Incorporation Modulates the Structure and Stability of Transthyretin in a Site-Specific Manner

Abnormal deposition of aggregated wild-type (WT) human transthyretin (TTR) and its pathogenic variants is responsible for cardiomyopathy and neuropathy related to TTR amyloidosis. The tryptophan (Trp) fluorescence measurements typically used to study structural changes of TTR do not yield site-specific information on the two Trp residues per TTR protomer. To obtain such information, tryptophan labeled with fluorine at the 5 and 6 positions (5FW and 6FW) was incorporated into TTR. Fluorescence of 5FW and 6FW-labeled WT-TTR (WT-5FW and WT-6FW) and a single-Trp mutant W41Y showed that the photophysics of incorporated fluoro-Trp is consistent with site-specific solvation of the indole ring of W41 and W79. ¹⁹F-NMR showed that solvent accessibility depends on both the location of the Trp and the position of the fluorine substituent in the indole ring. Unexpectedly, differences were observed in the rates of aggregation, with WT-6FW aggregating more rapidly than WT-5FW or WT-TTR. Real-time ¹⁹F-NMR urea unfolding experiments revealed that WT-5FW is kinetically more stable than WT-6FW, consistent with the aggregation assay. In addition, structural perturbations of residues distant from either Trp site are more extensive in WT-6FW. Notably, residues in the dimer interfaces are perturbed by 6FW at residue 79; pathogenic mutations in these regions are associated with reduced tetramer stability and amyloidogenesis. The differences in behavior that arise from the replacement of a fluorine at the 5-position of a tryptophan with one at the adjacent 6-position emphasize the delicate balance of stability in the TTR tetramer

Side-Chain Conformational Heterogeneity of Intermediates in the <i>Escherichia coli</i> Dihydrofolate Reductase Catalytic Cycle

<i>Escherichia coli</i> dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the χ₁ rotamer populations of the aromatic and γ-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of ³<i>J</i>_CγCO and ³<i>J</i>_CγN coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond time scale. This large volume of ³<i>J</i> data has allowed us to fit new Karplus parameterizations for aromatic side chains and to select the best available of previously determined parameters for Ile, Thr, and Val. The ³<i>J</i>_CγCO and ³<i>J</i>_CγN coupling constants are found to be extremely sensitive measures of side chain χ₁ rotamers and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand

Side Chain Conformational Averaging in Human Dihydrofolate Reductase

The three-dimensional structures of the dihydrofolate reductase enzymes from <i>Escherichia coli</i> (ecDHFR or ecE) and <i>Homo sapiens</i> (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and <i>E. coli</i> enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ₁ rotamer populations derived from measurements of ³<i>J</i>_CγCO and ³<i>J</i>_CγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ₁ rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ₁ rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side chain motions of critical residues

Probing the Non-Native H Helix Translocation in Apomyoglobin Folding Intermediates

Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)­amino]­ethyl}­amino)­naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C–L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state

Slow Dynamics of Tryptophan–Water Networks in Proteins

Water has a profound effect on the dynamics of biomolecules and governs many biological processes, leading to the concept that function is slaved to solvent dynamics within and surrounding the biomolecule. Protein conformational changes on μs–ms time scales are frequently associated with protein function, but little is known about the behavior of protein-bound water on these time scales. Here we have used NMR relaxation dispersion measurements to probe the tryptophan indoles in the enzyme dihydrofolate reductase (DHFR). We find that during structural changes on the μs–ms time scale, large chemical shift changes are often observed for the NH proton on the indole ring, while relatively smaller chemical shift changes are observed for the ring nitrogen atom. Comparison with experimental chemical shifts and density functional theory-based chemical shift predictions show that during the structural change the tryptophan indole NHs remain bound to water, but the geometry of the protein-bound water networks changes. These results establish that relaxation dispersion measurements can indirectly probe water dynamics and indicate that water can influence, or be influenced by, protein conformational changes on the μs–ms time scale. Our data show that structurally conserved bound water molecules can play a critical role in transmitting information between functionally important regions of the protein and provide evidence that internal protein motions can be coupled through the mediation of hydrogen-bonded water bound in the protein structure

Cofactor-Mediated Conformational Dynamics Promote Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway

The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm for the role of protein dynamics in enzyme catalysis. Previous studies have shown that the enzyme progresses through the kinetic cycle by modulating the dynamic conformational landscape in the presence of substrate dihydrofolate (DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP⁺). This study focuses on the quantitative description of the relationship between protein fluctuations and product release, the rate-limiting step of DHFR catalysis. NMR relaxation dispersion measurements of millisecond time scale motions for the E:THF:NADP⁺ and E:THF:NADPH complexes of wild-type and the Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded ground state and a low population of a closed state. The backbone structures of the occluded ground states of the wild-type and mutant proteins are very similar, but the rates of exchange with the closed excited states are very different. Integrated analysis of relaxation dispersion data and THF dissociation rates measured by stopped-flow spectroscopy shows that product release can occur by two pathways. The intrinsic pathway consists of spontaneous product dissociation and occurs for all THF-bound complexes of DHFR. The allosteric pathway features cofactor-assisted product release from the closed excited state and is utilized only in the E:THF:NADPH complexes. The L28F mutation alters the partitioning between the pathways and results in increased flux through the intrinsic pathway relative to the wild-type enzyme. This repartitioning could represent a general mechanism to explain changes in product release rates in other E. coli DHFR mutants

Structural Characterization of Interactions between the Double-Stranded RNA-Binding Zinc Finger Protein JAZ and Nucleic Acids

The interactions of the human double-stranded RNA-binding zinc finger protein JAZ with RNA or DNA were investigated using electrophoretic mobility-shift assays, isothermal calorimetry, and nuclear magnetic resonance spectroscopy. Consistent with previous reports, JAZ has very low affinity for duplex DNA or single-stranded RNA, but it binds preferentially to double-stranded RNA (dsRNA) with no detectable sequence specificity. The affinity of JAZ for dsRNA is unaffected by local structural features such as loops, overhangs, and bulges, provided a sufficient length of reasonably well-structured A-form RNA (about 18 bp for a single zinc finger) is present. Full-length JAZ contains four Cys₂His₂ zinc fingers (ZF1–4) and has the highest apparent affinity for dsRNA; two-finger constructs ZF12 and ZF23 have lower affinity, and ZF34 binds even more weakly. The fourth zinc finger, ZF4, has no measurable RNA-binding affinity. Single zinc finger constructs ZF1, ZF2, and ZF3 show evidence for multiple-site binding on the minimal RNA. Fitting of quantitative NMR titration and isothermal calorimetry data to a two-site binding model gave <i>K</i>_d1 ∼ 10 μM and <i>K</i>_d2 ∼ 100 μM. Models of JAZ–RNA complexes were generated using the high-ambiguity-driven biomolecular docking (HADDOCK) program. Single zinc fingers bind to the RNA backbone without sequence specificity, forming complexes with contacts between the RNA minor groove and residues in the N-terminal β strands and between the major groove and residues in the helix–kink–helix motif. We propose that the non-sequence-specific interaction between the zinc fingers of JAZ with dsRNA is dependent only on the overall shape of the A-form RNA

Mapping Interactions of the Intrinsically Disordered C‑Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR

The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), which are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using trans-intein splicing, we have generated full-length p53 constructs in which the C-terminal region is isotopically labeled with 15N for NMR analysis, allowing us to obtain atomic-level information on the C-terminal domains in the context of the full-length protein. Resonances of NLS and CTD residues have narrow linewidths, showing that these regions are largely solvent-exposed and dynamically disordered, whereas resonances from the folded TET are broadened beyond detection. Two regions of the CTD, spanning residues 369–374 and 381–388 and with high lysine content, make dynamic and sequence-independent interactions with DNA in regions that flank the p53 recognition element. The population of DNA-bound states increases as the length of the flanking regions is extended up to approximately 20 base pairs on either side of the recognition element. Acetylation of K372, K373, and K382, using a construct of the transcriptional coactivator CBP containing the TAZ2 and acetyltransferase domains, inhibits interaction of the CTD with DNA. This work provides high-resolution insights into the behavior of the intrinsically disordered C-terminal regions of p53 within the full-length tetramer and the molecular basis by which the CTD mediates DNA binding and specificity

Cofactor-Mediated Conformational Dynamics Promote Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway

The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm for the role of protein dynamics in enzyme catalysis. Previous studies have shown that the enzyme progresses through the kinetic cycle by modulating the dynamic conformational landscape in the presence of substrate dihydrofolate (DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP⁺). This study focuses on the quantitative description of the relationship between protein fluctuations and product release, the rate-limiting step of DHFR catalysis. NMR relaxation dispersion measurements of millisecond time scale motions for the E:THF:NADP⁺ and E:THF:NADPH complexes of wild-type and the Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded ground state and a low population of a closed state. The backbone structures of the occluded ground states of the wild-type and mutant proteins are very similar, but the rates of exchange with the closed excited states are very different. Integrated analysis of relaxation dispersion data and THF dissociation rates measured by stopped-flow spectroscopy shows that product release can occur by two pathways. The intrinsic pathway consists of spontaneous product dissociation and occurs for all THF-bound complexes of DHFR. The allosteric pathway features cofactor-assisted product release from the closed excited state and is utilized only in the E:THF:NADPH complexes. The L28F mutation alters the partitioning between the pathways and results in increased flux through the intrinsic pathway relative to the wild-type enzyme. This repartitioning could represent a general mechanism to explain changes in product release rates in other E. coli DHFR mutants

Structural Basis for Interaction of the Tandem Zinc Finger Domains of Human Muscleblind with Cognate RNA from Human Cardiac Troponin T

The human muscleblind-like proteins (MBNL) regulate tissue-specific splicing by targeting cardiac troponin T and other pre-mRNAs; aberrant targeting of CUG and CCUG repeat expansions frequently accompanies the neuromuscular disease myotonic dystrophy. We show, using biolayer interferometry (Octet) and NMR spectroscopy, that the zinc finger domains of MBNL isoform 1 (MBNL1) are necessary and sufficient for binding CGCU sequences within the pre-mRNA of human cardiac troponin T. Protein constructs containing zinc fingers 1 and 2 (zf12) and zinc fingers 3 and 4 (zf34) of MBNL1 each fold into a compact globular tandem zinc finger structure that participates in RNA binding. NMR spectra show that the stoichiometry of the interaction between zf12 or zf34 and the CGCU sequence is 1:1, and that the RNA is single-stranded in the complex. The individual zinc fingers within zf12 or zf34 are nonequivalent: the primary RNA binding surface is formed in each pair by the second zinc finger (zf2 or zf4), which interacts with the CGCU RNA sequence. The NMR structure of the complex between zf12 and a 15-base RNA of sequence ₉₅GUC­U<u>CG­CU</u>U­U­U­C­CCC₁₀₉, containing a single CGCU element, shows the single-stranded RNA wrapped around zf2 and extending to bind to the C-terminal helix. Bases C101, U102, and U103 make well-defined and highly ordered contacts with the protein, whereas neighboring bases are less well-ordered in the complex. Binding of the MBNL zinc fingers to cardiac troponin T pre-mRNA is specific and relatively simple, unlike the complex multiple dimer–trimer stoichiometries postulated in some previous studies