The role of protein complexes in human genetic disease.

Many human genetic disorders are caused by mutations in protein-coding regions of DNA. Taking protein structure into account has therefore provided key insight into the molecular mechanisms underlying human genetic disease. Although most studies have focused on the intramolecular effects of mutations, the critical role of the assembly of proteins into complexes is being increasingly recognized. Here, we review multiple ways in which consideration of protein complexes can help us to understand and explain the effects of pathogenic mutations. First, we discuss disorders caused by mutations that perturb intersubunit interactions in homomeric and heteromeric complexes. Second, we address how protein complex assembly can facilitate a dominant-negative mechanism, whereby mutated subunits can disrupt the activity of wild-type protein. Third, we show how mutations that change protein expression levels can lead to damaging stoichiometric imbalances. Finally, we review how mutations affecting different subunits of the same heteromeric complex often cause similar diseases, whereas mutations in different interfaces of the same subunit can cause distinct phenotypes.

Computational Modelling of Protein Complex Structure and Assembly.

Sequence and structure space are nowadays sufficiently large that we can use computational methods to model the structure of proteins based on sequence similarity alone. Not only useful as a standalone tool, homology modelling has also had a transformative effect on the ease with which we can solve crystal structures and electron density maps. Another technique-molecular dynamics-aims to model protein structures from first principles and, thanks to increases in computational power, is slowly becoming a viable tool for studying protein complexes. Finally, the prediction of protein assembly pathways from three-dimensional structures of complexes is also now becoming possible.

The genetic basis and evolution of red blood cell sickling in deer.

Crescent-shaped red blood cells, the hallmark of sickle-cell disease, present a striking departure from the biconcave disc shape normally found in mammals. Characterized by increased mechanical fragility, sickled cells promote haemolytic anaemia and vaso-occlusions and contribute directly to disease in humans. Remarkably, a similar sickle-shaped morphology has been observed in erythrocytes from several deer species, without obvious pathological consequences. The genetic basis of erythrocyte sickling in deer, however, remains unknown. Here, we determine the sequences of human ß-globin orthologues in 15 deer species and use protein structural modelling to identify a sickling mechanism distinct from the human disease, coordinated by a derived valine (E22V) that is unique to sickling deer. Evidence for long-term maintenance of a trans-species sickling/non-sickling polymorphism suggests that sickling in deer is adaptive. Our results have implications for understanding the ecological regimes and molecular architectures that have promoted convergent evolution of sickling erythrocytes across vertebrates.

Functional determinants of protein assembly into homomeric complexes.

Approximately half of proteins with experimentally determined structures can interact with other copies of themselves and assemble into homomeric complexes, the overwhelming majority of which (>96%) are symmetric. Although homomerisation is often assumed to a functionally beneficial result of evolutionary selection, there has been little systematic analysis of the relationship between homomer structure and function. Here, utilizing the large numbers of structures and functional annotations now available, we have investigated how proteins that assemble into different types of homomers are associated with different biological functions. We observe that homomers from different symmetry groups are significantly enriched in distinct functions, and can often provide simple physical and geometrical explanations for these associations in regards to substrate recognition or physical environment. One of the strongest associations is the tendency for metabolic enzymes to form dihedral complexes, which we suggest is closely related to allosteric regulation. We provide a physical explanation for why allostery is related to dihedral complexes: it allows for efficient propagation of conformational changes across isologous (i.e. symmetric) interfaces. Overall we demonstrate a clear relationship between protein function and homomer symmetry that has important implications for understanding protein evolution, as well as for predicting protein function and quaternary structure.

A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect.

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.

Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

Co-translational assembly of protein complexes.

The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

Two-photon absorption-molecular structure investigation using a porphycene chromophore with potential in photodynamic therapy.

Porphycenes have been shown to exhibit many advantageous properties when it comes to the application of two-photon absorption (TPA), a technique with potential use in the area of photodynamic therapy (PDT). A computational study of structure-reactivity relationships in the one- and two-photon absorption spectra of a series of 2,7,12,17-substituted porphycenes has been carried out using linear and quadratic density functional response theory. The focus has been on determining the effect on the spectra of electron donating and withdrawing substituents, the outcome of extending the conjugation lengths to these substituents, and the consequence of formation of metallo-porphycene complexes. In particular, we have looked at the use of TPA in order to improve the penetration depth of the therapeutic light dose, in terms of the position of the absorption maximum with respect to the optical window of tissue penetration, as well as the effect on the TPA cross section. The extent of conjugation was shown to be particularly crucial for increasing the TPA cross section, for both the electron withdrawing and donating substituents, while the inclusion of a metal in the center of the macrocycle was shown to benefit the absorption wavelength in terms of tissue penetration considerations.

Two-photon absorption in porphycenic macrocycles: the effect of tuning the core aromatic electronic structure.

Changing the core heteroatoms in porphycenic macrocycles can have a dramatic effect on the two-photon absorption properties via tuning of resonance enhancement between Q-band and Soret-band states.

Following the excited state relaxation dynamics of indole and 5-hydroxyindole using time-resolved photoelectron spectroscopy.

Time-resolved photoelectron spectroscopy was used to obtain new information about the dynamics of electronic relaxation in gas-phase indole and 5-hydroxyindole following UV excitation with femtosecond laser pulses centred at 249 nm and 273 nm. Our analysis of the data was supported by ab initio calculations at the coupled cluster and complete-active-space self-consistent-field levels. The optically bright (1)L(a) and (1)L(b) electronic states of (1)ππ∗ character and spectroscopically dark and dissociative (1)πσ∗ states were all found to play a role in the overall relaxation process. In both molecules we conclude that the initially excited (1)L(a) state decays non-adiabatically on a sub 100 fs timescale via two competing pathways, populating either the subsequently long-lived (1)L(b) state or the (1)πσ∗ state localised along the N-H coordinate, which exhibits a lifetime on the order of 1 ps. In the case of 5-hydroxyindole, we conclude that the (1)πσ∗ state localised along the O-H coordinate plays little or no role in the relaxation dynamics at the two excitation wavelengths studied.

Time resolved velocity map imaging of H-atom elimination from photoexcited imidazole and its methyl substituted derivatives.

The photoresistive properties of DNA bases, amino acids and corresponding subunits have received considerable attention through spectroscopic studies in recent years. One photoresistive property implicates the participation of (1)πσ* states, allowing electronically excited states to evolve either back to the electronic ground state or undergo direct dissociation along a heteroatom-hydride (X-H) coordinate. To this effect, time-resolved velocity map imaging (TR-VMI) studies of imidazole (a subunit of both adenine and histidine) and methylated derivatives thereof have been undertaken, with the goal of understanding the effects of increasing molecular complexity, through methylation, on the dynamics following photoexcitation at 200 nm. The results of these measurements clearly show that H-atom elimination along the N-H coordinate results in a bimodal distribution in the total kinetic energy release (TKER) spectra in both imidazole and it's methylated derivatives: 2-methyl, 4-methyl and 2,4-dimethylimidazole. The associated time constants for H-atoms eliminated with both high and low kinetic energies are all less than 500 fs. A noticeable increase in the time constants for the methylated derivatives is also observed. This could be attributed to either: ring methylation hindering in-plane and out-of-plane ring distortions which have been implicated as mediating excited state dynamics of these molecules or; an increase in the density of vibrational states at 200 nm causing an increased sampling of orthogonal modes, as opposed to modes which drive any dynamics that cause subsequent H-atom elimination. The results of these findings once again serve to illustrate the seemingly ubiquitous nature of (1)πσ* states in the photoexcited state dynamics of biomolecules and their subunits.