Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as $\alpha_1$-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create $\textit{conserpin}$, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.BTP is a Medical Research Council Career Development Fellow. AAN and JJH are supported by the Wellcome Trust (grant number WT 095195). SM acknowledges fellowship support from the Australian Research Council (FT100100960). NAB is an Australian Research Council Future Fellow (110100223). GIW is an Australian Research Council Discovery Outstanding Researcher Award Fellow (DP140100087). AMB is a National Health and Medical Research Senior Research Fellow (1022688). JCW is an NHMRC Senior Principal Research fellow and also acknowledges the support of an ARC Federation Fellowship. We thank the Australian Synchrotron for beam-time and technical assistance. This work was supported by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE) (www.massive.org.au). We acknowledge the Monash Protein Production Unit and Monash Macromolecular Crystallization Facilit

Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics

Identification of a rare p.G320R alpha-1-antitrypsin variant in emphysema and lung cancer patients

The alpha-1-antitrypsin (A1AT) gene is highly polymorphic, with more than 100 genetic variants identified of which some can affect A1AT protein concentration and/or function and lead to pulmonary and/or liver disease. This study reports on the characterization of a p.G320R variant found in two patients, one with emphysema and the other with lung cancer. This variant results from a single base-pair substitution in exon 4 of the A1AT gene, and has been characterized as P by isoelectric focusing. Functional evaluation of the A1AT p.G320R variant was through comparing specific trypsin inhibitory activity in two patients with pulmonary disorders, carriers of the p.G320R variant, and 19 healthy individuals, carriers of normal A1AT M variants. Results showed that specific trypsin inhibitory activity was lower in both emphysema (2.45 mU/g) and lung cancer (2.07 mU/g) patients than in carriers of the normal variants (range 2.51-3.71 mU/g). This rare A1AT variant is associated with reduced functional activity of A1AT protein. Considering that it was found in patients with severe pulmonary disorders, this variant could be of clinical significance

The YARHG Domain: An Extracellular Domain in Search of a Function

We have identified a new bacterial protein domain that we hypothesise binds to peptidoglycan. This domain is called the YARHG domain after the most highly conserved sequence-segment. The domain is found in the extracellular space and is likely to be composed of four alpha-helices. The domain is found associated with protein kinase domains, suggesting it is associated with signalling in some bacteria. The domain is also found associated with three different families of peptidases. The large number of different domains that are found associated with YARHG suggests that it is a useful functional module that nature has recombined multiple times

Quantitative sequence-function relationships in proteins based on gene ontology

<p>Abstract</p> <p>Background</p> <p>The relationship between divergence of amino-acid sequence and divergence of function among homologous proteins is complex. The assumption that homologs share function – the basis of transfer of annotations in databases – must therefore be regarded with caution. Here, we present a quantitative study of sequence and function divergence, based on the Gene Ontology classification of function. We determined the relationship between sequence divergence and function divergence in 6828 protein families from the PFAM database. Within families there is a broad range of sequence similarity from very closely related proteins – for instance, orthologs in different mammals – to very distantly-related proteins at the limit of reliable recognition of homology.</p> <p>Results</p> <p>We correlated the divergence in sequences determined from pairwise alignments, and the divergence in function determined by path lengths in the Gene Ontology graph, taking into account the fact that many proteins have multiple functions. Our results show that, among homologous proteins, the proportion of divergent functions decreases dramatically above a threshold of sequence similarity at about 50% residue identity. For proteins with more than 50% residue identity, transfer of annotation between homologs will lead to an erroneous attribution with a totally dissimilar function in fewer than 6% of cases. This means that for very similar proteins (about 50 % identical residues) the chance of completely incorrect annotation is low; however, because of the phenomenon of recruitment, it is still non-zero.</p> <p>Conclusion</p> <p>Our results describe general features of the evolution of protein function, and serve as a guide to the reliability of annotation transfer, based on the closeness of the relationship between a new protein and its nearest annotated relative.</p

Correlated Evolution of Nearby Residues in Drosophilid Proteins

Here we investigate the correlations between coding sequence substitutions as a function of their separation along the protein sequence. We consider both substitutions between the reference genomes of several Drosophilids as well as polymorphisms in a population sample of Zimbabwean Drosophila melanogaster. We find that amino acid substitutions are “clustered” along the protein sequence, that is, the frequency of additional substitutions is strongly enhanced within ≈10 residues of a first such substitution. No such clustering is observed for synonymous substitutions, supporting a “correlation length” associated with selection on proteins as the causative mechanism. Clustering is stronger between substitutions that arose in the same lineage than it is between substitutions that arose in different lineages. We consider several possible origins of clustering, concluding that epistasis (interactions between amino acids within a protein that affect function) and positional heterogeneity in the strength of purifying selection are primarily responsible. The role of epistasis is directly supported by the tendency of nearby substitutions that arose on the same lineage to preserve the total charge of the residues within the correlation length and by the preferential cosegregation of neighboring derived alleles in our population sample. We interpret the observed length scale of clustering as a statistical reflection of the functional locality (or modularity) of proteins: amino acids that are near each other on the protein backbone are more likely to contribute to, and collaborate toward, a common subfunction

Automated functional classification of experimental and predicted protein structures

BACKGROUND: Proteins that are similar in sequence or structure may perform different functions in nature. In such cases, function cannot be inferred from sequence or structural similarity. RESULTS: We analyzed experimental structures belonging to the Structural Classification of Proteins (SCOP) database and showed that about half of them belong to multi-functional fold families for which protein similarity alone is not adequate to assign function. We also analyzed predicted structures from the LiveBench and the PDB-CAFASP experiments and showed that accurate homology-based functional assignments cannot be achieved approximately one third of the time, when the protein is a member of a multi-functional fold family. We then conducted extended performance evaluation and comparisons on both experimental and predicted structures using our Functional Signatures from Structural Alignments (FSSA) algorithm that we previously developed to handle the problem of classifying proteins belonging to multi-functional fold families. CONCLUSION: The results indicate that the FSSA algorithm has better accuracy when compared to homology-based approaches for functional classification of both experimental and predicted protein structures, in part due to its use of local, as opposed to global, information for classifying function. The FSSA algorithm has also been implemented as a webserver and is available at

Analysis of the Peptidoglycan Hydrolase Complement of Lactobacillus casei and Characterization of the Major γ-D-Glutamyl-L-Lysyl-Endopeptidase

Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties

Local Function Conservation in Sequence and Structure Space

We assess the variability of protein function in protein sequence and structure space. Various regions in this space exhibit considerable difference in the local conservation of molecular function. We analyze and capture local function conservation by means of logistic curves. Based on this analysis, we propose a method for predicting molecular function of a query protein with known structure but unknown function. The prediction method is rigorously assessed and compared with a previously published function predictor. Furthermore, we apply the method to 500 functionally unannotated PDB structures and discuss selected examples. The proposed approach provides a simple yet consistent statistical model for the complex relations between protein sequence, structure, and function. The GOdot method is available online (http://godot.bioinf.mpi-inf.mpg.de)