Solution properties of γ‐crystallins: Hydration of fish and mammal γ‐crystallins

Lens γ crystallins are found at the highest protein concentration of any tissue, ranging from 300 mg/mL in some mammals to over 1000 mg/mL in fish. Such high concentrations are necessary for the refraction of light, but impose extreme requirements for protein stability and solubility. γ‐crystallins, small stable monomeric proteins, are particularly associated with the lowest hydration regions of the lens. Here, we examine the solvation of selected γ‐crystallins from mammals (human γD and mouse γS) and fish (zebrafish γM2b and γM7). The thermodynamic water binding coefficient B₁ could be probed by sucrose expulsion, and the hydrodynamic hydration shell of tightly bound water was probed by translational diffusion and structure‐based hydrodynamic boundary element modeling. While the amount of tightly bound water of human γD was consistent with that of average proteins, the water binding of mouse γS was found to be relatively low. γM2b and γM7 crystallins were found to exhibit extremely low degrees hydration, consistent with their role in the fish lens. γM crystallins have a very high methionine content, in some species up to 15%. Structure‐based modeling of hydration in γM7 crystallin suggests low hydration is associated with the large number of surface methionine residues, likely in adaptation to the extremely high concentration and low hydration environment in fish lenses. Overall, the degree of hydration appears to balance stability and tissue density requirements required to produce and maintain the optical properties of the lens in different vertebrate species.     

Cooperative hydrophobic core interactions in the β‐trefoil architecture

Symmetric protein architectures have a compelling aesthetic that suggests a plausible evolutionary process (i.e., gene duplication/fusion) yielding complex architecture from a simpler structural motif. Furthermore, symmetry inspires a practical approach to computational protein design that substantially reduces the combinatorial explosion problem, and may provide practical solutions for structure optimization. Despite such broad relevance, the role of structural symmetry in the key area of hydrophobic core‐packing cooperativity has not been adequately studied. In the present report, the threefold rotational symmetry intrinsic to the β‐trefoil architecture is shown to form a geometric basis for highly‐cooperative core‐packing interactions that both stabilize the local repeating motif and promote oligomerization/long‐range contacts in the folding process. Symmetry in the β‐trefoil structure also permits tolerance towards mutational drift that involves a structural quasi‐equivalence at several key core positions.     

A rapid fluorogenic GPCR–β‐arrestin interaction assay

Detection of protein–protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein‐coupled receptor (GPCR)–β‐arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal‐to‐noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease‐activated receptor‐1 activation developed in minutes and required specific serine–threonine residues in the receptor carboxyl tail, consistent with a classical G protein‐coupled receptor kinase dependent β‐arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.     

Solution properties of γ‐crystallins: Compact structure and low frictional ratio are conserved properties of diverse γ‐crystallins

γ‐crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, γ‐crystallins have high contents of sulfur‐containing methionine and cysteine, but this reaches extremes in fish γM‐crystallins with up to 15% Met. In addition, fish γM‐crystallins do not conserve the paired tryptophan residues found in each domain in mammalian γ‐crystallins and in the related β‐crystallins. To gain insight into important, evolutionarily conserved properties and functionality of γ‐crystallins, zebrafish (Danio rerio) γM2b and γM7 were compared with mouse γS and human γD. For all four proteins, far UV CD spectra showed the expected β‐sheet secondary structure. Like the mammalian proteins, γM7 was highly soluble but γM2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full‐length and truncated versions of human αB‐crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse γ‐crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of γ‐crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.     

Conserved buried water molecules enable the β‐trefoil architecture

Available high‐resolution crystal structures for the family of β‐trefoil proteins in the structural databank were queried for buried waters. Such waters were classified as either: (a) unique to a particular domain, family, or superfamily or (b) conserved among all β‐trefoil folds. Three buried waters conserved among all β‐trefoil folds were identified. These waters are related by the threefold rotational pseudosymmetry characteristic of this protein architecture (representing three instances of an identical structural environment within each repeating trefoil‐fold motif). The structural properties of this buried water are remarkable and include: residing in a cavity space no larger than a single water molecule, exhibiting a positional uncertainty (i.e., normalized B‐factor) substantially lower than the average Cα atom, providing essentially ideal H‐bonding geometry with three solvent‐inaccessible main chain groups, simultaneously serving as a bridging H‐bond for three different β‐strands at a point of secondary structure divergence, and orienting conserved hydrophobic side chains to form a nascent core‐packing group. Other published work supports an interpretation that these interactions are key to the formation of an efficient folding nucleus and folded thermostability. The fundamental threefold symmetric structural element of the β‐trefoil fold is therefore, surprisingly, a buried water molecule.     

β‐Bulges: Extensive structural analyses of β‐sheets irregularities

β‐Sheets are quite frequent in protein structures and are stabilized by regular main‐chain hydrogen bond patterns. Irregularities in β‐sheets, named β‐bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β‐strands. They are implicated in protein‐protein interactions and are introduced to avoid β‐strand aggregation. Five different types of β‐bulges are defined. Previous studies on β‐bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β‐bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β‐bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574–1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β‐bulges are preferably localized at the N‐ and C‐termini of β‐strands, but contrary to the earlier studies, no significant conservation of β‐bulges was observed among structural homologues. Displacement of β‐bulges along the sequence was also investigated by Molecular Dynamics simulations.     

Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3′-to-5′ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in k_cat/K_m in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure–function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.     

Rules for connectivity of secondary structure elements in protein: Two–layer αβ sandwiches

In protein structures, the fold is described according to the spatial arrangement of secondary structure elements (SSEs: α‐helices and β‐strands) and their connectivity. The connectivity or the pattern of links among SSEs is one of the most important factors for understanding the variety of protein folds. In this study, we introduced the connectivity strings that encode the connectivities by using the types, positions, and connections of SSEs, and computationally enumerated all the connectivities of two‐layer αβ sandwiches. The calculated connectivities were compared with those in natural proteins determined using MICAN, a nonsequential structure comparison method. For 2α‐4β, among 23,000 of all connectivities, only 48 were free from irregular connectivities such as loop crossing. Of these, only 20 were found in natural proteins and the superfamilies were biased toward certain types of connectivities. A similar disproportional distribution was confirmed for most of other spatial arrangements of SSEs in the two‐layer αβ sandwiches. We found two connectivity rules that explain the bias well: the abundances of interlayer connecting loops that bridge SSEs in the distinct layers; and nonlocal β‐strand pairs, two spatially adjacent β‐strands located at discontinuous positions in the amino acid sequence. A two‐dimensional plot of these two properties indicated that the two connectivity rules are not independent, which may be interpreted as a rule for the cooperativity of proteins.     

Surface enhanced Raman spectroscopy distinguishes amyloid Β‐protein isoforms and conformational states

Amyloid β‐protein (Aβ) self‐association is one process linked to the development of Alzheimer's disease (AD). Aβ peptides, including its most abundant forms, Aβ40 and Aβ42, are associated with the two predominant neuropathologic findings in AD, vascular and parenchymal amyloidosis, respectively. Efforts to develop therapies for AD often have focused on understanding and controlling the assembly of these two peptides. An obligate step in these efforts is the monitoring of assembly state. We show here that surface‐enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) readily distinguishes Aβ40 and Aβ42. We show further, through comparison of assembly dependent changes in secondary structure and morphology, that the SERS/PCA approach unambiguously differentiates closely related assembly stages not readily differentiable by circular dichroism spectroscopy, electron microscopy, or other techniques. The high discriminating power of SERS/PCA is based on the rich structural information present in its spectra, which comprises not only on interatomic resonances between covalently associated atoms and hydrogen bond interactions important in controlling secondary structure, but effects of protein orientation relative to the substrate surface. Coupled with the label‐free, single molecule sensitivity of SERS, the approach should prove useful for determining structure activity relationships, suggesting target sites for drug development, and for testing the effects of such drugs on the assembly process. The approach also could be of value in other systems in which assembly dependent changes in protein structure correlate with the formation of toxic peptide assemblies.     

Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this β7–β8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by β7–β8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.     

Intrinsic secondary structure propensities of the amino acids,using statistical ϕ–ψ matrices: Comparison with experimental scales

Today there are several different experimental scales for the intrinsic α-helix as well as β-strand, propensities of the 20 amino acids obtained from the thermodynamic analysis of various model systems. These scales do not compare well with those extracted from statistical analysis of three-dimensional structure databases. Possible explanations for this could be the limited size of the databases used, the definitions of intrinsic propensities, or the theoretical approach. Here we report a statistical determination of α-helix and β-strand propensities derived from the analysis of a database of 279 three-dimensional structures. Contrary to what has been generally done, we have considered a particular residue as in α-helix or β-strand conformation by looking only at its dihedral angles (?–ψ matrices). Neither the identity nor the conformation of the surrounding residues in the amino acid sequence has been taken into consideration. Pseudoenergy empirical scales have been calculated from the statistical propensities. These scales agree very well with the experimental ones in relative and absolute terms. Moreover, its correlation with the average of the experimental scales for α-helix or β-strand is as good as the correlations of the individual experimental scales with the average. These results show that by using a large enough database and a proper definition for the secondary structure propensities, it is possible to obtain a scale as good as any of experimental origin. Interestingly the ?–ψ analysis of the Ramachandran plot suggests that the amino acids could have different β-strand propensities in different subregions of the β-strand area. © 1994 Wiley-Liss, Inc.     

Oligomerization of a symmetric β‐trefoil protein in response to folding nucleus perturbation

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl‐terminal region of the (internally located) FN of the de novo designed purely‐symmetric β‐trefoil protein Symfoil‐4P. This mutation involves wholesale replacement of a repeating trefoil‐fold motif with a “blade” motif from a β‐propeller protein, and postulated to trap that region of the Symfoil‐4P FN in a nonproductive folding intermediate. The resulting protein (termed “Bladefoil”) is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.     

Kinetic study of β‐amyloid residue accessibility using reductive alkylation and mass spectrometry

Beta‐amyloid peptide (Aβ) is the major protein constituent found in senile plaques in Alzheimer's disease (AD). It is believed that Aβ plays a role in neurodegeneration associated with AD and that its toxicity is related to its structure or aggregation state. In this study, an approach based on chemical modification of primary amines and mass spectrometric (MS) detection was used to identify residues on Aβ peptide that were exposed or buried upon changes in peptide structure associated with aggregation. Results indicate that the N terminus was the most accessible primary amine in the fibril, followed by lysine 28, then lysine 16. A kinetic analysis of the data was then performed to quantify differences in accessibility between these modification sites. We estimated apparent equilibrium unfolding constants for each modified site of the peptide, and determined that the unfolding constant for the N terminus was approximately 100 times greater than that for K28, which was about six times greater than that for K16. Understanding Aβ peptide structure at the residue level is a first step in designing novel therapies for prevention of Aβ structural transitions and/or cell interactions associated with neurotoxicity in Alzheimer's disease. Biotechnol. Bioeng. 2009; 104: 181–192 © 2009 Wiley Periodicals, Inc.     

Ab initio folding of a trefoil‐fold motif reveals structural similarity with a β‐propeller blade motif

Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain‐swapped arrangement at the interface of the N‐ and C‐termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N‐ and C‐termini in the overall architecture; however, the folding requirement of the primordial motif is poorly understood, and tolerance to circular permutation is essentially unknown. The β‐trefoil protein fold is a threefold‐symmetric architecture where the repeating ~42‐mer “trefoil‐fold” motif assembles via a domain‐swapped arrangement. The trefoil‐fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β‐trefoil trimeric assembly. The trefoil‐fold sequence is not predicted to adopt the trefoil‐fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact “blade” motif from the β‐propeller architecture. Expression of a trefoil‐fold sequence and circular permutants shows that only the wild‐type N‐terminal motif definition yields an intact β‐trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil‐fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil‐fold structural features, but is more structurally homologous to a β‐propeller blade motif.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β‐barrel membrane proteins using Opa₆₀ from Neisseria gonorrhoeae as a model system. Opa₆₀ is an eight‐stranded β‐barrel with long extracellular loops (～63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa₆₀ in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β‐barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β‐barrel. The removal of the loop resonances significantly improved the assignment of the Opa₆₀ β‐barrel region (97% of the resonances corresponding to the β‐barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full‐length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β‐barrel membrane proteins.     

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

α‐Crystallin is a multimeric eye lens protein having molecular chaperone‐like function which is crucial for lens transparency. The stability and unfolding‐refolding properties of α‐crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native‐like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α‐crystallin revealed dry native‐like core of α‐crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol^?1 on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol^?1 on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α‐crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 549–560, 2014.     

Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix

Aurora‐A regulates the recruitment of TACC3 to the mitotic spindle through a phospho‐dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora‐A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora‐A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical‐repeat region of CHC, not a recognized phospho‐reader domain. This potentially widespread mechanism of phospho‐recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.     

A comparison of convergently evolved insect silks that share β‐sheet molecular structure

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately β‐sheet with lesser amounts of β‐turns, unordered and α‐helical protein also detected. The orientation of the β‐sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel β‐sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 630–639, 2014.