Fast parallel proteolysis (FASTpp) is a method to determine the thermostability of proteins by measuring which fraction of protein resists rapid proteolytic digestion. [1]
Fast parallel proteolysis (FASTpp) is a method to determine the thermostability of proteins by measuring which fraction of protein resists rapid proteolytic digestion. [1]
Proteolysis is widely used in biochemistry and cell biology to probe protein structure. [2] [3] In "limited trypsin proteolysis", low amounts of protease digest both folded and unfolded protein but at largely different rates: unstructured proteins are cut more rapidly, while structured proteins are cut at a slower rate (sometimes by orders of magnitude). Recently, several other assays of protein stability based on proteolysis have been proposed, exploiting other proteases with high specificity for cleaving unfolded proteins. These include Pulse Proteolysis, [4] Proteolytic Scanning Calorimetry [5] and FASTpp.
FASTpp measures the quantity of protein that resists digestion under various conditions. To this end, a thermostable protease is used, which cleaves specifically at exposed hydrophobic residues. The FASTpp assay combines the thermal unfolding, specificity of a thermostable protease for the unfolded fraction with the separation power of SDS-PAGE. [6] Due to this combination, FASTpp can detect changes in the fraction folded over a large physico-chemical range of conditions including temperatures up to 85 °C, pH 6–9, presence or absence of the whole proteome. Applications range from biotechnology to study of point mutations and ligand binding assays.
FASTpp has been used to probe: [1]
First, a cell lysate is generated by glass beads beating, pressure homogenisation or chemical or physical lysis methods that do not denature the protein(s) of interest. (Optionally for targeted analysis) a protein of interest is purified out of this lysate by affinity methods based on intrinsically disordered tags [12] or other suitable purification strategies, often involving several orthogonal chromatographic steps.
This (total or purified) protein solution is aliquoted into several tubes of a PCR strip. All aliquots are exposed in parallel in a thermal gradient PCR cycler to different maximal temperatures in presence of the thermostable protease thermolysin (see figure). Automated temperature control is achieved in a thermal gradient cycler (commonly used for PCRs). Reaction products can be separated by SDS-PAGE or western blot. [6] The protease thermolysin can be fully inactivated by EDTA. This feature of thermolysin makes FASTpp compatible with subsequent trypsin digestion e.g. for mass spectrometry. [13] [14] [7]
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion.
Protein folding is the physical process where a protein chain is translated into its native three-dimensional structure, typically a "folded" conformation, by which the protein becomes biologically functional. Via an expeditious and reproducible process, a polypeptide folds into its characteristic three-dimensional structure from a random coil. Each protein exists first as an unfolded polypeptide or random coil after being translated from a sequence of mRNA into a linear chain of amino acids. At this stage, the polypeptide lacks any stable three-dimensional structure. As the polypeptide chain is being synthesized by a ribosome, the linear chain begins to fold into its three-dimensional structure.
A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in numerous biological pathways, including digestion of ingested proteins, protein catabolism, and cell signaling.
Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers – specifically polypeptides – formed from sequences of amino acids, which are the monomers of the polymer. A single amino acid monomer may also be called a residue, which indicates a repeating unit of a polymer. Proteins form by amino acids undergoing condensation reactions, in which the amino acids lose one water molecule per reaction in order to attach to one another with a peptide bond. By convention, a chain under 30 amino acids is often identified as a peptide, rather than a protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations driven by a number of non-covalent interactions, such as hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, cryo-electron microscopy (cryo-EM) and dual polarisation interferometry, to determine the structure of proteins.
A metalloproteinase, or metalloprotease, is any protease enzyme whose catalytic mechanism involves a metal. An example is ADAM12 which plays a significant role in the fusion of muscle cells during embryo development, in a process known as myogenesis.
In genetics, a missense mutation is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution.
In molecular biology, an intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure, typically in the absence of its macromolecular interaction partners, such as other proteins or RNA. IDPs range from fully unstructured to partially structured and include random coil, molten globule-like aggregates, or flexible linkers in large multi-domain proteins. They are sometimes considered as a separate class of proteins along with globular, fibrous and membrane proteins.
In materials science and molecular biology, thermostability is the ability of a substance to resist irreversible change in its chemical or physical structure, often by resisting decomposition or polymerization, at a high relative temperature.
Gamma secretase is a multi-subunit protease complex, itself an integral membrane protein, that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. Proteases of this type are known as intramembrane proteases. The most well-known substrate of gamma secretase is amyloid precursor protein, a large integral membrane protein that, when cleaved by both gamma and beta secretase, produces a short 37-43 amino acid peptide called amyloid beta whose abnormally folded fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients. Gamma secretase is also critical in the related processing of several other type I integral membrane proteins, such as Notch, ErbB4, E-cadherin, N-cadherin, ephrin-B2, or CD44.
Thermolysin is a thermostable neutral metalloproteinase enzyme produced by the Gram-positive bacteria Bacillus thermoproteolyticus. It requires one zinc ion for enzyme activity and four calcium ions for structural stability. Thermolysin specifically catalyzes the hydrolysis of peptide bonds containing hydrophobic amino acids. However thermolysin is also widely used for peptide bond formation through the reverse reaction of hydrolysis. Thermolysin is the most stable member of a family of metalloproteinases produced by various Bacillus species. These enzymes are also termed 'neutral' proteinases or thermolysin -like proteinases (TLPs).
Κ-casein, or kappa casein, is a mammalian milk protein involved in several important physiological processes. Chymosin splits K-casein into an insoluble peptide and water-soluble glycomacropeptide (GMP). GMP is responsible for an increased efficiency of digestion, prevention of neonate hypersensitivity to ingested proteins, and inhibition of gastric pathogens. The human gene for κ-casein is CSN3.
ATP-dependent Clp protease proteolytic subunit (ClpP) is an enzyme that in humans is encoded by the CLPP gene. This protein is an essential component to form the protein complex of Clp protease.
In molecular biology short linear motifs (SLiMs), linear motifs or minimotifs are short stretches of protein sequence that mediate protein–protein interaction.
Astacins are a family of multidomain metalloendopeptidases which are either secreted or membrane-anchored. These metallopeptidases belong to the MEROPS peptidase family M12, subfamily M12A. The protein fold of the peptidase domain for members of this family resembles that of thermolysin, the type example for clan MA and the predicted active site residues for members of this family and thermolysin occur in the motif HEXXH.
TopFIND is the Termini oriented protein Function Inferred Database (TopFIND) is an integrated knowledgebase focused on protein termini, their formation by proteases and functional implications. It contains information about the processing and the processing state of proteins and functional implications thereof derived from research literature, contributions by the scientific community and biological databases.
Terminal amine isotopic labeling of substrates (TAILS) is a method in quantitative proteomics that identifies the protein content of samples based on N-terminal fragments of each protein and detects differences in protein abundance among samples.
A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes.
Bacillolysin is an enzyme. This enzyme catalyses the following chemical reaction
NanoDSF is a modified differential scanning fluorimetry method to determine protein stability employing intrinsic tryptophan or tyrosine fluorescence.
Chemoproteomics entails a broad array of techniques used to identify and interrogate protein-small molecule interactions. Chemoproteomics complements phenotypic drug discovery, a paradigm that aims to discover lead compounds on the basis of alleviating a disease phenotype, as opposed to target-based drug discovery, in which lead compounds are designed to interact with predetermined disease-driving biological targets. As phenotypic drug discovery assays do not provide confirmation of a compound's mechanism of action, chemoproteomics provides valuable follow-up strategies to narrow down potential targets and eventually validate a molecule's mechanism of action. Chemoproteomics also attempts to address the inherent challenge of drug promiscuity in small molecule drug discovery by analyzing protein-small molecule interactions on a proteome-wide scale. A major goal of chemoproteomics is to characterize the interactome of drug candidates to gain insight into mechanisms of off-target toxicity and polypharmacology.