Protein-induced Conformational Changes Research Articles

NMR Titration Studies in Z-DNA Dynamics.

Chemical shift perturbation (CSP) is a simple NMR technique for studying the DNA binding of proteins. Titration of the unlabeled DNA into the 15N-labeled protein is monitored by acquiring a two-dimensional (2D) heteronuclear single-quantum correlation (HSQC) spectrum at each step of the titration. CSP can also provide information on the DNA-binding dynamics of proteins, as well as protein-induced conformational changes in DNA. Here, we describe the titration of DNA for the 15N-labeled Z-DNA-binding protein, monitored via 2D HSQC spectra. NMR titration data can be analyzed with the active B-Z transition model to provide the protein-induced B-Z transition dynamics of DNA.

Single-Molecule Fluorescence Methods to Study Protein-RNA Interactions Underlying Biomolecular Condensates.

Many biomolecular condensates, including nucleoli and stress granules, form via dynamic multivalent protein-protein and protein-RNA interactions. These molecular interactions nucleate liquid-liquid phase separation (LLPS) and determine condensate properties, such as size and fluidity. Here, we outline the experimental procedures for single-molecule fluorescence experiments to probe protein-RNA interactions underlying LLPS. The experiments include single-molecule Förster (Fluorescence) resonance energy transfer (smFRET) to monitor protein-induced conformational changes in the RNA, protein-induced fluorescence enhancement (PIFE) to measure protein-RNA encounters, and single-molecule nucleation experiments to quantify the association and buildup of proteins on a nucleating RNA. Together, these experiments provide complementary approaches to elucidate a molecular view of the protein-RNA interactions that drive ribonucleoprotein condensate formation.

Investigating the Role of Msh4-Msh5 ATPASE Activity During Homologous Recombination

Msh4-Msh5 or MutSgamma is a protein belonging to the MutS family of DNA repair proteins that are mainly involved in post-replicative mismatch repair for the purpose of maintaining genome integrity. Msh4-Msh5 has a different role in which the protein facilitates crossover formation during meiotic recombination in many eukaryotic organisms. Failure to form crossovers results in improper segregation of chromosomes during meiosis, which can lead to infertility and birth defects. Understanding the structural and functional intricacies of this protein will help to elucidate how it functions in crossover formation and meiotic recombination. Previously, our lab has shown that Msh4-Msh5 binds to Holliday junctions (HJ) and HJ-like structures such as 3’ overhangs, single-stranded forks, and D-loops with high affinity, as opposed to DNA duplexes to which the protein binds with much weaker affinity. Generally, our FRET measurements show Msh4-Msh5 displaces the single strand in single-strand containing substrates and induces stacked junction-like structures for substrates more closely resembling Holliday junctions. We are investigating how the presence of nucleotides affects the DNA binding properties of S. cerevisiae Msh4-Msh5. Our approach uses a combination of fluorescently-labeled substrates and nucleotides to determine dissociation constants in the presence of different recombination intermediates while also examining protein-induced conformational changes. We are pairing these results with kinetic studies of ATP hydrolysis in the presence of the same DNA recombination intermediates to gain a clearer understanding of how Msh4-Msh5 ATPase activity is coupled with DNA and protein conformational changes. Through this work, we are investigating the potential molecular switch mechanism of Msh4-Msh5 by which other members of the MutS family have been show to function, while comparing our results from studying S. cerevisiae Msh4-Msh5 to previous results reporting on human Msh4-Msh5.

Probing RNA-Protein Interactions and RNA Compaction by Sedimentation Velocity Analytical Ultracentrifugation.

Recent advances in multi-wavelength analytical ultracentrifugation (MWL-AUC) combine the power of an exquisitely sensitive hydrodynamic-based separation technique with the added dimension of spectral separation. This added dimension has opened up new doors to much improved characterization of multiple, interacting species in solution. When applied to structural investigations of RNA, MWL-AUC can precisely report on the hydrodynamic radius and the overall shape of an RNA molecule by enabling precise measurements of its sedimentation and diffusion coefficients and identify the stoichiometry of interacting components based on spectral decomposition. Information provided in this chapter will allow an investigator to design experiments for probing ion and/or protein-induced global conformational changes of an RNA molecule and exploit spectral differences between proteins and RNA to characterize their interactions in a physiological solution environment.

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

Calmodulin is a prototypical and versatile Ca(2+) sensor with EF hands as its high-affinity Ca(2+) binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca(2+)-dependent signaling. Upon binding Ca(2+), calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulin's ability to bind Ca(2+). Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca(2+)-bound calmodulin but show different sensitivity to Ca(2+) for their activation. Protein crystal structures and other experiments show that, depending on which SK2 splice variant it binds to, calmodulin adopts drastically different conformations with different affinities for Ca(2+) at its C-lobe. Such target protein-induced conformational changes make calmodulin a dynamic Ca(2+) sensor capable of responding to different Ca(2+) concentrations in cellular Ca(2+) signaling.

DEAD-box protein facilitated RNA folding in vivo

In yeast mitochondria the DEAD-box helicase Mss116p is essential for respiratory growth by acting as group I and group II intron splicing factor. Here we provide the first structure-based insights into how Mss116p assists RNA folding in vivo. Employing an in vivo chemical probing technique, we mapped the structure of the ai5γ group II intron in different genetic backgrounds to characterize its intracellular fold. While the intron adopts the native conformation in the wt yeast strain, we found that the intron is able to form most of its secondary structure, but lacks its tertiary fold in the absence of Mss116p. This suggests that ai5γ is largely unfolded in the mss116-knockout strain and requires the protein at an early step of folding. Notably, in this unfolded state misfolded substructures have not been observed. As most of the protein-induced conformational changes are located within domain D1, Mss116p appears to facilitate the formation of this largest domain, which is the scaffold for docking of other intron domains. These findings suggest that Mss116p assists the ordered assembly of the ai5γ intron in vivo.

14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3)

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the alpha-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G(alpha) subunit. Since RGS proteins interact with G(alpha) subunits through their RGS domains, it is reasonable to assume that the 14-3-3 protein can either sterically occlude the G(alpha) interaction surface of RGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein binding affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two single-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Our measurements revealed that the 14-3-3 protein binding induces structural changes in both the N-terminal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolated RGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 protein in a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 was solved at 2.3A resolution. The data obtained from the resolution of the structure of the RGS domain suggest that the 14-3-3 protein-induced conformational change affects the region within the G(alpha)-interacting portion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity of RGS3.

Platform for in situ real-time measurement of protein-induced conformational changes of DNA

A platform for in situ and real-time measurement of protein-induced conformational changes in dsDNA is presented. We combine electrical orientation of surface-bound dsDNA probes with an optical technique to measure the kinetics of DNA conformational changes. The sequence-specific Escherichia coli integration host factor is utilized to demonstrate protein-induced bending upon binding of integration host factor to dsDNA probes. The effects of probe surface density on binding/bending kinetics are investigated. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed electrodes, making it amenable for use as a high throughput assay.

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex.

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the beta(2) adrenergic receptor (beta(2)AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta(2)AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy.

Linear Response of Biomolecules To External Perturbations: Revisit Induce-fit

Atoms in proteins, viewed as interlinked hubs, communicate with each other in complying with governed physical forces. The deviations of their positions from the mean, known as fluctuations, are essential in mediating functionally relevant biological processes maintaining one's daily life. The coupled fluctuations between pairs of atoms, the fluctuation covariance, can be determined analytically by Normal Mode Analysis, or numerically by MD simulations. Our study considers how this network of atoms reacts in response to external perturbations. Effects of such perturbations are exemplified by ligand- or (another) protein-induced conformational changes as well as the appreciated structural distortion of crystalline structures from its solution conformers. We assume the response of the system has linear departure from the mean under small perturbations on the Hamiltonian at equilibrium state. The formulated linear response theories, either time-dependent or -independent [1], says that the positional change of a given atom i is the accumulative sum of fluctuation covariance ij, at unperturbed state, multiplied by the force exerted on atom j. The time-dependent response function determined from MD simulation of carbonmonoxy myoglobin is used to track time-dependent conformational changes upon photo-dissociation of CO. The consequently obtained understanding of perturbation propagation is compared with experimental results. Also, the structural distortion of X-ray-characterized ubiquitin from its solution conformer can be well explained by the induce-fit theory, in a wider sense, while population-shift does not account for such a deviation.[1] Ikeguchi M, Ueno J, Sato M, and Kidera A. (2005) Phys Rev Lett, 94, 078102.

Protein-induced Conformational Changes of RNA During the Assembly of Human Signal Recognition Particle

The human signal recognition particle (SRP) is a large RNA–protein complex that targets secretory and membrane proteins to the endoplasmic reticulum membrane. The S domain of SRP is composed of roughly half of the 7SL RNA and four proteins (SRP19, SRP54, and the SRP68/72 heterodimer). In order to understand how the binding of proteins induces conformational changes of RNA and affects subsequent binding of other protein subunits, we have performed chemical and enzymatic probing of all S domain assembly intermediates. Ethylation interference experiments show that phosphate groups in helices 5, 6 and 7 that are essential for the binding of SRP68/72 are all on the same face of the RNA. Hydroxyl radical footprinting and dimethylsulphate (DMS) modifications show that SRP68/72 brings the lower part of helices 6 and 8 closer. SRP68/72 binding also protects the SRP54 binding site (helix 8 asymmetric loop) from chemical modification and RNase cleavage, whereas, in the presence of both SRP19 and SRP68/72, the long strand of helix 8 asymmetric loop becomes readily accessible to chemical and enzymatic probes. These results indicate that the RNA platform observed in the crystal structure of the SRP19–SRP54M–RNA complex already exists in the presence of SRP68/72 and SRP19. Therefore, SRP68/72, together with SRP19, rearranges the 7SL RNA in an SRP54 binding competent state.

Ss-LrpB from Sulfolobus solfataricus Condenses about 100 Base Pairs of Its Own Operator DNA into Globular Nucleoprotein Complexes

Ss-LrpB from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2 is a member of the Lrp-like family of Bacterial/Archaeal transcription regulators that binds its own control region at three regularly spaced and partially conserved 15-bp-long imperfect palindromes. We have used atomic force microscopy to analyze the architecture of Ss-LrpB.DNA complexes with a different stoichiometry formed with the wild type operator and with an operator mutant. Binding of dimeric Ss-LrpB to all three target sites is accompanied by the formation of globular complexes, in which the protein induces strong DNA deformations. Furthermore, DNA contour length foreshortening of these complexes indicates DNA wrapping, with about 100 bp being condensed. The average bending angle is 260 degrees . The establishment of protein-protein contacts between Ss-LrpB dimers in these globular complexes will contribute to the cooperativity of the binding. The profound remodeling of the control region is expected to have a strong impact on gene expression and might constitute the key element in the autoregulatory process.

Novel tethered particle motion analysis of CI protein-mediated DNA looping in theregulation of bacteriophage lambda

The tethered particle motion (TPM) technique has attracted great interest because of itssimplicity and the wealth of information that it can provide on protein-induced conformationalchanges in nucleic acids. Here we present an approach to TPM methodology and analysisthat increases the efficiency of data acquisition and facilitates interpretation of TPMassays. In particular, the statistical analysis that we propose allows fast data processing,minimal data selection and visual display of the distribution of molecular behaviour. Themethodology proved useful in verifying CI protein-mediated DNA looping in bacteriophageλ and in differentiating between two different types of loops, stable and dynamic, whoserelative occurrence seems to be a function of the distance between the operators as well astheir relative angular orientation. Furthermore, the statistical analysis indicates that CIbinding per se slightly shortens the DNA.

Folding of the td pre-RNA with the help of the RNA chaperone StpA.

The td group I intron is inserted in the reading frame of the thymidylate synthase gene, which is mainly devoid of structural elements. In vivo, translation of the pre-mRNA is required for efficient folding of the intron into its splicing-competent structure. The ribosome probably resolves exon-intron interactions that interfere with splicing. Uncoupling splicing from translation, by introducing a non-sense codon into the upstream exon, reduces the splicing efficiency of the mutant pre-mRNA. Alternatively to the ribosome, co-expression of genes that encode proteins with RNA chaperone activity promote folding of the td pre-mRNA in vivo. These proteins also efficiently accelerate folding of the td pre-mRNA in vitro. In order to understand the mechanism of action of RNA chaperones, we probed the impact of the RNA chaperone StpA on the structure of the td intron in vivo and compared it with that of the well characterized Cyt-18 protein, which is a group-I-intron-specific splicing factor. We found that the two proteins have opposite effects on the structure of the td intron. While StpA loosens the three-dimensional structure, Cyt-18 tightens it up. Furthermore, mutations that destabilize the intron structure render the mutants sensitive to StpA, whereas splicing of these mutants is rescued by Cyt-18. Our results provide direct evidence for protein-induced conformational changes within a catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the stabilization of the native three-dimensional structure.

RNA chaperone StpA loosens interactions of the tertiary structure in the td group I intron in vivo

Efficient splicing of the td group I intron in vivo is dependent on the ribosome. In the absence of translation, the pre-mRNA is trapped in nonnative-splicing-incompetent conformations. Alternatively, folding of the pre-mRNA can be promoted by the RNA chaperone StpA or by the group I intron-specific splicing factor Cyt-18. To understand the mechanism of action of RNA chaperones, we probed the impact of StpA on the structure of the td intron in vivo. Our data suggest that StpA loosens tertiary interactions. The most prominent structural change was the opening of the base triples, which are involved in the correct orientation of the two major intron core domains. In line with the destabilizing activity of StpA, splicing of mutant introns with a reduced structural stability is sensitive to StpA. In contrast, Cyt-18 strengthens tertiary contacts, thereby rescuing splicing of structurally compromised td mutants in vivo. Our data provide direct evidence for protein-induced conformational changes within catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the overall compactness of the td intron in vivo.

Radiosensitivity of DNA in a specific protein-DNA complex: the lac repressor- lac operator complex

Purpose : To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor- lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor. Materials and methods : Two structures of the complex were extracted from the PDB databank: crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4' and H5') to OH • and of the FSB probabilities, along: (1) DNA in the complex; (2) DNA in the complex depleted of the repressor; and (3) a linear DNA having the same sequence. An 80bp fragment bearing the operator was irradiated alone or in presence of the repressor. The relative probabilities of FSB at each nucleotide were determined using sequencing gel electrophoresis. Results : Calculations predict modulation of the accessibility of H4' and H5' atoms and of the probabilities of FSB along the DNA fragments of complexes. This is due to the protein-induced conformational change and to masking by bound protein. The best agreement with the experimental FSB was observed for calculations that use the crystallography-based structure. Conclusions : For specific DNA-protein complexes, our calculations can predict the protein radiolytic footprints on DNA. They show the significant contribution of the protein-induced DNA conformational change to DNA protection.

Conformational Changes of the BS2 Operator DNA upon Complex Formation with the Antennapedia Homeodomain Studied by NMR with13C/15N-labeled DNA

The NMR structures have been determined for a13C/15N doubly labeled 14 base-pair DNA duplex comprising the BS2 operator sequence both free in solution and in the complex with theAntennapedia homeodomain. The impact of the DNA labeling is assessed from comparison with a previous structure of the same complex that was determined using isotope labeling only for the protein. Differences between the two structure determinations are nearly completely limited to the DNA, which retains the global B -conformation of the free DNA also in the complex. Local protein-induced conformational changes are a narrowing of the minor groove due to the interaction with the N-terminal arm of the homeodomain, and changes of the sugar puckers of the deoxyriboses G5 and C6, which are apparently induced by van der Waals interactions with Tyr25, and with Gln50 and Arg53, respectively. The high conservation of these amino acid residues in homeodomains suggests that protein-induced shifts in some sugar puckers contribute to the affinity of homeodomains to their cognate DNA. The data obtained here with the Antennapedia homeodomain-DNA complex clearly show that nucleic acid isotope-labeling can support detailed conformational characterization of DNA in complexes with proteins, which will be indispensable for structure determinations of complexes containing globally distorted DNA conformations.

Cooperative assembly of signal recognition particle RNA with protein SRP19.

Signal recognition particle (SRP) is a ribonucleoprotein complex involved in the targeting of secretory proteins to the lipid bilayer of the endoplasmic reticulum. SRP contains the protein SRP19, which is an important structural and functional component, believed to promote the assembly of the particle. We have purified the human SRP19 protein to homogeneity from recombinant bacteria which overexpress the polypeptide, and have studied details of the binding to SRP RNA via gel mobility shift and RNase sensitivity assays. SRP19 interacts with two SRP RNA conformers with different affinities such that the more compact RNA species is bound more avidly. Furthermore, binding was found to be highly cooperative. Binding constants and Hill coefficients were determined for several mutant SRP RNAs in which individual RNA helices were deleted. These results confirmed that both SRP RNA helices 6 and 8 are important for SRP19 binding. Enzymatic RNA structure probing of a 150-nucleotide mutant SRP RNA fragment and of the corresponding RNA-SRP19 complex showed that cooperativity may be due to protein-induced conformational changes in the large domain of the SRP RNA. Finally, SRP19 bound specifically not only to SRP RNA but also to the A-form of Escherichia coli 5S ribosomal RNA, thereby indicating structural similarities between these two RNA molecules.

Binding of an HIV Rev peptide to Rev responsive element RNA induces formation of purine-purine base pairs.

The Rev responsive element (RRE) is an RNA secondary structural element within the env gene of HIV and is the binding site for the viral Rev protein. Formation of the Rev-RRE complex is involved in regulation of splicing and transport of mRNA from the nucleus. To understand the structural basis for the specific recognition of RRE by Rev, we have studied a model system for this interaction using NMR. We have obtained a specific 1:1 complex between an RNA derived from stem IIB of RRE, which contains the highest affinity Rev binding site, and a modified Rev34-50 peptide, which binds the RRE as an alpha-helix [Tan, R., et al. (1993) Cell 73, 1031-1040]. Binding of the peptide was accompanied by a conformational change in the RNA, which resulted in the formation of additional base pairs not present in the free RNA. Two of these induced base pairs are purine-purine pairs within the internal loop of RRE, which had been previously proposed on the basis of biochemical experiments [Bartel, D.P., et al. (1991) Cell 67, 529-536]. The formation of non-Watson-Crick base pairs, interactions in the major groove, and protein-induced conformational changes may prove to be common characteristics of RNA recognition of proteins.