Intramolecular Communication Research Articles

Intramolecular electronic communication in ferrocene-based β-diketonato copper(II) complexes as observed by an electrochemical study

The reaction of [Cu(OAc)2.H2O]2 (1) with β-diketones FcCOCH2COR (R=CF3, 2a, CH3, 2b, Ph=C6H5, 2c, and Fc=Fe(η5–C5H4)(η5–C5H5), 2d), afforded in a straightforward synthetic methodology a series of ferrocenyl-functionalized β-diketonato copper(II) complexes 3a–3d of general formula [Cu(FcCOCHCOR)2]. The environment around copper(II) is square planar with the ferrocenyl moieties being positioned opposite to each other. All complexes display in their cyclic voltammograms resolved and electrochemical reversible ferrocenyl oxidations in the range 98⩽Eo′⩽420mV versus FcH/FcH+. An electrochemical and chemical irreversible Cu(II) reduction was observed in the range −1624⩽Epc⩽−1102mV. Good intramolecular communication between neutral ferrocenyl and oxidised, positively charged ferrocenium cations explained the well resolved ferrocenyl redox processes. The observed intramolecular communication was quantified with the linear equation Eo′=98.5ΣχR−629 which relates formal ferrocenyl oxidation potentials with the sum of R-group electronegativities, ΣχR. It was possible to show that in [Cu(FcCOCHCOFc)2], 3d, one ferrocenyl group on each β-diketonato ligand is first oxidized before the second ferrocenyl group on the same β-diketonato ligand is oxidized. In a spectro-electrochemical study, an intervalent charge transfer band (IVCT) could be detected for the mixed valent intermediates of 3d that are electrochemically generated. For complex 3a, only the normal ligand to metal charge transfer band at ca. 650nm was observed. This demonstrate that communication between Fc and Fc+ groups are more effective within a specific β-diketonato ligand, such as (Fc+COCHCOFc) in 3d, than across a copper centre, [Fc+COCHCOR)Cu(FcCOCHCOR)], presumably because of the longer distance between the Fc⋯Fc+ species across the copper centre than within a single ligand.

Intramolecular communication and electrochemical observation of the 17-electron ruthenocenium cation in fluorinated ruthenocene-containing β-diketones; polymorphism of C10H21 and C10F21 derivatives

Ruthenocene-containing β-diketones, RcCOCH2COR with Rc = ruthenocenyl and R = C10F21 (1), CF3 (2), C6F5 (3), C10H21 (4), CH3 (5) and C6H5 (6), were synthesised by Claisen condensation of the appropriate methyl ester with acetylruthenocene, and their spectroscopic, electrochemical and thermal properties were compared. A new synthetic route utilising 1,2,3-benzotriazol-1-ylethanone (9) or 1,2,3-benzotriazol-1-yl(ruthenocenyl)methanone (10) as a reactant, rather than the conventional esters, was found to be more efficient for β-diketone synthesis. The apparent acid dissociation constants, pKa′, of the new ruthenocene-containing β-diketones are 7.14(4) (1, R = C10F21), 9.92(3) (3, R = C6F5) and 10.06(2) (4, R = C10H21). Peak anodic potentials of the ruthenocenyl group of 1–6, pKa′ values and the FTIR ν(CO) stretching frequencies of the precursor esters, RCOOCH3, correlated linearly with the Gordy scale group electronegativity, χR, of the C10F21 (χC10F21 = 3.04), C6F5 (χC6F5 = 2.46), C10H21 (χC10H21 = 2.43) and other R-groups. An electrochemical study in the non-interacting solvent and electrolyte system CH2Cl2/0.1 mol dm−3 [N(nBu4)][B(C6F5)4] revealed electrochemically irreversible one-electron transfer Rc/Rc+ couples in the potential range 650 90% abundance under the conditions of study and the first order rate constant of enol to keto conversion varied between 220 and 50 000 s−1 depending on solvent (CDCl3 or CD3CN) and R-groups. Thermal analysis (DSC) of 1 and 4 showed no liquid crystalline mesophase behaviour but definite polymorphism was observed. β-diketones 1 and 4 exist as low temperature polymorphs below 42 °C or 12 °C respectively. The high temperature polymorphs converted to isotropic liquids at 83 °C (compound 1) or 52 °C (compound 4).

Elucidation of Calcium Ion and Phospholipid Binding Profiles of Multiple Dysferlin Isoforms

Dysferlin is a large type II peripheral membrane protein associated with the sarcolemma and T-tubular system of muscle fibers. It contains seven C2 domains and two DysF domains and has been implicated in membrane repair. This repair is thought to be mediated through calcium-triggered exocytosis of intracellular vesicles, which leads to the formation of a membrane patch. Due to dysferlin's structural similarity to synaptotagmin1 it is assumed that its role in the overall scheme of membrane repair is to act as a calcium and membrane sensor. The C2A domain is the first of the seven C2 domains and is assumed to be the protein's main calcium and membrane sensor.The C2A domain exists in nature as a combination of two different isoforms derived from alternative readings of the first exon sequence: the canonical C2A (WT) isoform derived from exon 1, and the C2Av1 (V1) isoform derived from exon 1'. The two different isoforms of the C2A domain were studied to probe the calcium and lipid binding affinities of dysferlin. Using isothermal titration calorimetry (ITC) the calcium and lipid binding profiles of the two constructs were established. These profiles indicate a difference in the phospholipid dependence of calcium binding between the two constructs as well as a uniform calcium dependence on the phospholipid binding. Initial approximations of the constructs' binding parameters were estimated individually and can be fit globally using a partition function approach.By determining dysferlin's binding profile we can better characterize its function in the scheme of membrane repair, as well as form a better understanding of the intramolecular communication that tunes its physiological response.

α,ω‐Di([5]trovacenyl) Sulfides TVC–S n –TVC ( n = 1–4) and TVC–SCH 2 S–TVC: a Study in Intramolecular Communication

The oligosulfur-bridged dinuclear paramagnetic complexes di([5]trovacenyl) sulfide (3··), di([5]trovacenyl) trisulfide (5··), di([5]trovacenylthio)methane (6··), and di([5]trovacenyl) tetrasulfide (7··) have been prepared by the metathesis reactions of [5]trovacenyllithium with SCl2, and [5]lithiotrovacenylthiolate (2·) with SCl2, CH2Cl2, and S2Cl2, respectively, in which 2· was obtained by the reaction of [5]trovacenyllithium with 1 equiv. of elemental sulfur at ambient temperature. Combined with the previously reported di([5]trovacenyl) disulfide (4··), these complexes have been systematically investigated by means of cyclic voltammetry, electron paramagnetic resonance (EPR) spectroscopy, magnetic susceptometry, and/or X-ray diffraction analysis, which reveal weak antiferromagnetic spin–spin exchange interactions. The monosulfur-bridged 3··, in particular, displays the strongest intramolecular spin–spin exchange interaction of |JEPR| = 2.8 cm–1 and Jχ = –1.39 cm–1. This study also indicates that with an increasing number of sulfur atoms in the bridge, the intramolecular communication of biradicals throught the 1D saturated sulfur atom bridge significantly weakens both in solution and in the solid state compared to those of unsaturated carbon-based bridges.

Mechanisms of Intramolecular Communication in a Hyperthermophilic Acylaminoacyl Peptidase: A Molecular Dynamics Investigation

Protein dynamics and the underlying networks of intramolecular interactions and communicating residues within the three-dimensional (3D) structure are known to influence protein function and stability, as well as to modulate conformational changes and allostery. Acylaminoacyl peptidase (AAP) subfamily of enzymes belongs to a unique class of serine proteases, the prolyl oligopeptidase (POP) family, which has not been thoroughly investigated yet. POPs have a characteristic multidomain three-dimensional architecture with the active site at the interface of the C-terminal catalytic domain and a β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In the present contribution, protein dynamics signatures of a hyperthermophilic acylaminoacyl peptidase (AAP) of the prolyl oligopeptidase (POP) family, as well as of a deletion variant and alanine mutants (I12A, V13A, V16A, L19A, I20A) are reported. In particular, we aimed at identifying crucial residues for long range communications to the catalytic site or promoting the conformational changes to switch from closed to open ApAAP conformations. Our investigation shows that the N-terminal α1-helix mediates structural intramolecular communication to the catalytic site, concurring to the maintenance of a proper functional architecture of the catalytic triad. Main determinants of the effects induced by α1-helix are a subset of hydrophobic residues (V16, L19 and I20). Moreover, a subset of residues characterized by relevant interaction networks or coupled motions have been identified, which are likely to modulate the conformational properties at the interdomain interface.

Allosteric modulators can restore function in an amino acid neurotransmitter receptor by slightly altering intra-molecular communication pathways.

Mutations, even if not directly in the ligand binding sites of proteins, can lead to disease. In cell surface receptors, this can happen if they uncouple conformational changes that take place upon agonist (or antagonist) binding to the extracellular domain and the intracellular response. Uncoupling can take place by disrupting a major allosteric propagation pathway between the extra- and intracellular domains. Here I provide a mechanistic explanation: I first describe how propagation takes place; second, what can happen in the presence of a disease-related mutation which is distant from the binding site; and finally, how drugs may overcome this disruption and rescue function. The mutations in the glycine receptor α1 subunit (α1R271Q/L) which cause the neuromotor disorder hyperekplexia are on example of such allosteric mutations. In this issue of the BJP, Shan et al. show that normal function was restored to these mutant receptors by substitution of the segment which contained the mutated position, by a homologous one. An allosteric drug could mimic the effects of such substitution. Within this framework, I highlight the advantages of allosteric drugs and the challenges in their design.

Electrochemical Illumination of Intramolecular Communication in Ferrocene-Containingtris-β-Diketonato Aluminum(III) Complexes; Cytotoxicity of Al(FcCOCHCOCF3)3

The series of ferrocene-containing tris-β-diketonato aluminum(III) complexes [Al(FcCOCHCOR)(3)] (R = CF(3), 1; CH(3), 2; C(6)H(5), 3; and Fc = ferrocenyl = Fe(η(5)-C(5)H(5))(η(5)-C(5)H(4)), 4) were synthesized and investigated structurally and electrochemically; complex 1 was subjected to cytotoxicity tests. (1)H NMR-spectroscopy distinguished between the mer and fac isomers of 2 and 3. Complex 1 existed only as the mer isomer. A single crystal X-ray crystallographic determination of the structure of a mer-isomer of Al(FcCOCHCOCF(3))(3), 1, (Z = 4, space group P2(1)2(1)2(1)) demonstrated extensive delocalization of all bonds which explained the pronounced electrochemically observed intramolecular communication between molecular fragments. In contrast to electrochemical studies in CH(2)Cl(2)/[N((n)Bu)(4)][PF(6)], the use of the supporting electrolyte [N((n)Bu)(4)][B(C(6)F(5))(4)] allowed identification of all Fc/Fc(+) electrochemical couples by cyclic and square wave voltammetry for 1-4. For R = Fc, formal reduction potentials of the six ferrocenyl groups were found to be E°' = 33, 123, 304, 432, 583, and 741 mV versus free ferrocene respectively. Complex 1 (IC(50) = 10.6 μmol dm(-3)) was less cytotoxic than the free FcCOCH(2)COCF(3) ligand having IC(50) = 6.8 μmol dm(-3) and approximately 2 orders of magnitude less toxic to human HeLa neoplastic cells than cisplatin (IC(50) = 0.19 μmol dm(-3)).

Binding-folding induced regulation of AF1 transactivation domain of the glucocorticoid receptor by a cofactor that binds to its DNA binding domain.

Intrinsically disordered (ID) regions of proteins commonly exist within transcription factors, including the N-terminal domain (NTD) of steroid hormone receptors (SHRs) that possesses a powerful activation function, AF1 region. The mechanisms by which SHRs pass signals from a steroid hormone to control gene expression remain a central unresolved problem. The role of N-terminal activation function AF1, which exists in an intrinsically disordered (ID) conformation, in this process is of immense importance. It is hypothesized that under physiological conditions, ID AF1 undergoes disorder/order transition via inter- and intra-molecular communications, which allows AF1 surfaces to interact with specific co-regulatory proteins, critical for the final outcome of target gene expression regulated by SHRs. However, the means by which AF1 acquires functionally folded conformations is not well understood. In this study, we tested whether binding of jun dimerization protein 2 (JDP2) within the DNA binding domain (DBD) of the glucocorticoid receptor (GR) leads to acquisition of functionally active structure in its AF1/NTD. Our results show that signals mediated from GR DBD:JDP2 interactions in a two domain GR fragment, consisting of the entire NTD and little beyond DBD, significantly increased secondary/tertiary structure formation in the NTD/AF1. This increased structure formation facilitated AF1’s interaction with specific co-regulatory proteins and subsequent glucocorticoid response element-mediated AF1 promoter:reporter activity. These results support the hypothesis that inter- and intra-molecular signals give a functionally active structure(s) to the GR AF1, which is important for its transcriptional activity.

Change in Allosteric Network Affects Binding Affinities of PDZ Domains: Analysis through Perturbation Response Scanning

The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs) and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS) that couples elastic network models with linear response theory (LRT) to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain). With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) dynamics plays a key role in allosteric regulations of PDZs, (ii) the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii) this might be the mechanism that each PDZ uses to tailor their binding specificities regulation.

Heat Maps for Intramolecular Communication in an RNP Enzyme Encoding Glutamine

Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNA(Gln) complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 Å, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code.

Loop 5-directed Compounds Inhibit Chimeric Kinesin-5 Motors: IMPLICATIONS FOR CONSERVED ALLOSTERIC MECHANISMS

The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.

Detailed Tuning of Structure and Intramolecular Communication Are Dispensable for Processive Motion of Myosin VI

Dimeric myosin VI moves processively hand-over-hand along actin filaments. We have characterized the mechanism of this processive motion by measuring the impact of structural and chemical perturbations on single-molecule processivity. Processivity is maintained despite major alterations in lever arm structure, including replacement of light chain binding regions and elimination of the medial tail. We present kinetic models that can explain the ATP concentration-dependent processivities of myosin VI constructs containing either native or artificial lever arms. We conclude that detailed tuning of structure and intramolecular communication are dispensable for processive motion, and further show theoretically that one proposed type of nucleotide gating can be detrimental rather than beneficial for myosin processivity.

A Molecular Switch between the Outer and the Inner Vestibules of the Voltage-gated Na+ Channel

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K+ channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na+ channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNaV1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state (“ultra-slow inactivation,” IUS) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of IUS. The fact that mutations at site 1575 modulate IUS produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.

Dynein at odd angles?

Cytoplasmic dynein drives vesicular transport from the periphery to the cell body of neurons. Missense mutations in the dynein tail domain cause neurodegenerative disease in mouse models. new data on the effect of one such dynein mutation provide insight into the intramolecular communication and flexible stepping of this essential cellular motor.

A molecular switch between the outer and the inner vestibules of the voltage-gated Na+ channel

Background Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na channels but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid I1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the rNaV1.4 channel may act as molecular switch allowing for interaction between outer and inner vestibule.

Conserved amino acids participate in the structure networks deputed to intramolecular communication in the lutropin receptor.

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) particularly susceptible to spontaneous pathogenic gain-of-function mutations. Protein structure network (PSN) analysis on wild-type LHR and two constitutively active mutants, combined with in vitro mutational analysis, served to identify key amino acids that are part of the regulatory network responsible for propagating communication between the extracellular and intracellular poles of the receptor. Highly conserved amino acids in the rhodopsin family GPCRs participate in the protein structural stability as network hubs in both the inactive and active states. Moreover, they behave as the most recurrent nodes in the communication paths between the extracellular and intracellular sides in both functional states with emphasis on the active one. In this respect, non-conservative loss-of-function mutations of these amino acids is expected to impair the most relevant way of communication between activating mutation sites or hormone-binding domain and G protein recognition regions.

Homodinuclear f–f and Heterodinuclear f–p Lanthanide Helicates

AbstractLanthanide helicates are of interest due to their ability to emit either in the visible or in the NIR region. In this context it is important to prepare heterodinuclear helicates and to use them for the investigation of intramolecular communication processes between different complex units. In here we describe the ligand synthesis and self‐assembly of homo‐ and heterodinuclear helicates based on β‐diketonate, catechol‐acyl hydrazone and 2‐amido‐8‐hydroxyquinoline ligands. Strongly emitting homodinuclear europium(III) complexes are obtained in addition to heterodinuclear coordination compounds. Some of the latter exhibit intramolecular energy transfer behaviour with internal quenching of an aluminium trisquinolinate and enhancement of the NIR emission at an ytterbium(III), neodymium(III) or erbium(III) complex unit.

TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation

Transcription of eukaryotic mRNA encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (GTFs; TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter leading to preinitiation complex (PIC) formation and transcription1. In TFIID-dependent activation pathways, this TATA box Binding Protein (TBP)-containing GTF is first recruited on the promoter through interaction with activators1-3 and cooperates with TFIIA to form a committed PIC4. However, neither the mechanisms by which activation signals are communicated between these factors, nor the structural organization of the activated PIC are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer-promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TBP within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator binding site and the proximal promoter region, and this loop is topologically locked by a TFIIA-Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer-promoter communication and provide important new structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.

Mechanism of GTPase-Activity-Induced Self-Assembly of Human Guanylate Binding Protein 1

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of large GTPases (LGs). In the course of GTP hydrolysis, the protein undergoes structural changes leading to self-assembly of the protein, which is a characteristic property of all family members. For self-assembly, the protein employs two distinct interaction sites, one of which is located within the LG domain of the protein located at the N-terminus, and the second is located in the C-terminal α-helical domain. Here, we identify intramolecular contacts between the LG domain and the helical part of hGBP1, which relay nucleotide-dependent structural changes from the N-terminus to the C-terminus and thereby mediate tetramer formation of the protein through a second contact site at the C-terminus. Furthermore, we demonstrate the impact of this intramolecular communication on the enzymatic activity of hGBP1 and on its cellular localization.

Structural insights into retinitis pigmentosa from unfolding simulations of rhodopsin mutants

Disease-causing missense mutations in membrane proteins, such as rhodopsin mutations associated with the autosomal dominant form of retinitis pigmentosa (ADRP), are often linked to defects in folding and/or trafficking. The mechanical unfolding of wild-type rhodopsin was compared with that of 20 selected ADRP-linked mutants more or less defective in folding and retinal binding. Rhodopsin fold is characterized by networks of amino acids in the retinal and G-protein binding sites likely to play a role in the stability and function of the protein. The distribution of highly connected nodes in the network reflects the existence of a diffuse intramolecular communication inside and between the 2 poles of the helix bundle, which makes pathogenic mutations share similar phenotypes irrespective of topological and physicochemical differences between them. Because of this communication, the ADRP-linked rhodopsin mutations share a more or less marked ability to impair selected hubs in the protein structure network. The extent of this structural effect relates to the severity of the biochemical defect caused by mutation. The investigative strategy employed in this study is likely to apply to all structurally known membrane proteins particularly susceptible to misassembly-causing mutations.