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Analysis of amino acid composition of secondary structural elements branching at the b-carbon tends to destabilize an a-helix (Val, Thr, Ile) due to steric clashes, these aa are better suited to b-sheets where they stand out of the chain Ser, Asp, Asn disrupt a-helices due to H-bonding donor/acceptors sites near the main chain where they compete for N-H or C=O Pro tends to disrupt both a-helices and b-strands due to its ring structure Gly readily fits into every sorts of structural motifs because of its small size predictions of secondary structure of 6 or fewer residues, taking the above (and other) considerations into account, proved to be ~60-70% accurate; reasons include the not abrupt change of preference one aa has for one structural motif or another tertiary interactions may push the same peptide to adopt another secondary structure in a different local environment many sequences can adopt alternative conformations in different proteins the VDLLKN sequence shown in purple assumes a helical or a b-strand conformation in two different proteins (3WRP. pdb and 2HLA.pdb) Folds protein tertiary structures are divided into five main classes according to the secondary structure content of their domains: all-a domains, all-b domains (b-barrels, e.g. Greek key motif), a+b domains (irregular fashion of arrangement), a/b domains (b-a-b motifs) and “others” each class contains many different folds further classified into families no necessary functional connection is in this type of classification: a certain type of function is often, but not always, restricted to a certain type of fold (convergent evolution) – fold of a protein is “only” a scaffold to which functions (active sites and different binding sites) are “added” a protein of 100 aa may have 20100 possible sequences/conformations, but an estimate says that there are only ~1000 folds in nature (we know a few hundreds of them so far); if we consider only the # of human genes (even without splice variants) there should be more than ~30,000 individual conformations if all protein sequences would adopt a new structure conformation is more conserved than sequence similar folds may have very low sequence identity and that is true vica versa, nevertheless, ~30% sequence identity generally means similar structure (may have diverged from a common ancestor; homology modeling of proteins is based on this premise) modules: genetically mobile units manifested as separate protein domains, functional units that are shared by proteins of similar functions; there are domain (fold) families throughout the phylogenetic tree to deliver similar functions (small differences in sequence with evolutionary conserved regions featured in multiple sequence alignment of protein primary structures) some folds are more favored than others as they represent a more stable structure and some proteins may converge towards these folds over the course of evolution; a number of folds though are found in only one group of proteins the CATH domain database classifies domains into approximately 800 fold families, ten of these folds are highly populated and are referred to as 'super-folds‘; super-folds are defined as folds for which there are at least three structures without significant sequence similarity (the most populated is the α/β-barrel super-fold) b-barrels large b-sheet that twists and coils to form a closed structure first and last b-strands are H-bonded typical antiparallel arrangement of strands found in proteins spanning membranes (e.g. porins) and in proteins that bind hydrophobic ligands inside the barrel (e.g. in a lipocalin fold) CATH (Class, Architecture, Topology, Homologous) database Similar database: SCOP (Structural Classification Of Proteins) all known protein structures are sorted according to their folds The Anfinsen experiment Christian Anfinsen: Ribonuclease A (RNaseA; single polypeptide chain, 124 aa, 4 S-S bonds) structure was disrupted by 8 M urea (carbamide) and b-SH-EtOH (both reversibly). 8 M Urea (or 6 M guanidinium chloride) resolve H-bonds while b-SH-EtOH reduces S-S bonds to sulfhydryls (cysteines). if reoxidized while still in 8 M urea and then got rid of urea (by dialysis) only 1% of activity was regained (wrong disulfides formed pairs in urea) 105 different ways of pairing 8 –SH and only one is the native, active RNase A Driving force: decrease of free energy the 104 wrong conformations spontaneously converted into the active conformation in the presence of trace amounts of b-SH-EtOH (in ~10 hours!) Denaturation of proteins denaturating agents have chaotropic properties and disrupt the 3D structure of proteins (or DNA/RNA) chaotropic agents interfere with intramolecular H-bonding and van der Waals forces (hydrophobic interactions) and denature biomacromolecules chaotropes also break down the H-bonded network of H2O allowing proteins more structural freedom and encouraging extension and denaturation Examples of chaotropic agents: 6-8 M urea, 2 M thiourea, 6 M guanidinium chloride, 4.5 M LiClO4 and in general high generic salt concentrations can also exhibit chaotropic effects: they shield electronic charges preventing stabilization of salt bridges and also weaken H-bridges which are more stable in less polar media (not being completely solvated at high concentration, ions interact with dipoles of H-binding partners, which is more favorable than H-bridging itself); they also perturb solubility of proteins taking H2O out of the hydration sphere of proteins – (reversible) precipitation/fractionation of proteins opposite of chaotropes (disorder-maker, destabilizer) are kosmotropes (order-maker, stabilizer): they stabilize proteins in solution, increase structuring of water molecules SO42-, HPO42-, Mg2+ , Ca2+ , Li+, Na+, H+, OH- and HPO42- (small ions with high charge density) are good kosmotropes exhibiting stronger interactions with H2O than H2O with itself and therefore capable of breaking H2O-H2O H-bonds; non-ionic kosmotropes: trehalose, glucose, proline, terc-butanol SCN-, H2PO4-, HSO4-, HCO3-, I-, Cl-, NO3-, NH4+, Cs+, K+, (NH2)3C+ (guanidinium) and (CH3)4N+ (tetramethylammonium) ions are rather chaotropes proteins are most stable in solution when surrounded by fully H-bonded H2O as H2O with spare H-bonding capacity has higher entropy and is more “aggressive”; such reactive H2O behaves in a similar way to raising T that denatures proteins optimum stabilization of biological macromolecule by salt requires a mixture of a kosmotropic anion with a chaotropic cation and the chaotropic ions (with their weak aqueous interactions) should be the direct counterions to the protein and the kosmotropic ions (with their strong aqueous interactions) in the bulk; (NH ) SO is a good salt for stabilizing protein structure/activity when the anion and cation have similar affinities for H2O they are able to remove H2O from each other most easily, to become ion-paired. A small ion of high charge density plus a large counter-ion of low charge density forms a highly soluble, solvent-separated hydrated but clustered ion pair as the large ion cannot break through its counter-ion's hydration shell (for example, CaI2, AgF and LiI versus CaF2, AgI) Hofmeister series of ions precipitating proteins: (Franz Hofmeister was also the one who proposed first in 1902 that amino acids build up proteins via peptide bonds (even before Emil Fischer)) true when proteins are of net negative charge, pH>pI, may reverse if pH<pI, different counterion or pH is present in the original experiment they used a mixture of egg white proteins, did not control pH and ovalbumin was of negative charge and they got the following series: anions: citrate3- > SO42- = tartrate2- > HPO42- > CrO42- > acetate- > HCO3- > Cl- > NO3- > ClO3cations: Mg2+ > Li+ > Na+ = K+ > NH4+ (reversible) Salting out/precipitation of proteins based on smaller solubility of proteins at high salt concentration critical concentration varies for different proteins (fractionation/purification of proteins, e.g. albumins vs. globulins) used also to concentrate proteins from dilute solutions (e.g. after gel filtration [size-exclusion chromatography]) done generally by (solid) (NH4)2SO4 (final concentration expressed as the % of the saturated (NH4)2SO4 solution) followed by filtration or centrifugation dissolution in appropriate buffer and dialysis is used to remove high salt concentrations afterwards and get the protein dissolved back again Caution: some ions first increase the solubility of a protein (salting in) while others may permanently denature/precipitate/poison certain proteins or enzymes (e.g. heavy metal poisoning – irreversible complexation occurs) protein “salting out” results from interfacial effects of strongly hydrated anions near the protein surface so removing water molecules from the protein solvation sphere and dehydrating the surface protein “salting in” results from protein-counter ion binding and the consequently higher net protein charge and solvation; it occurs where the protein has little net charge near its pI primarily by weakly hydrated anions. protein solubility is minimal at the pI (net charge is zero), below or above charged protein molecules repel each other resulting in better solubility precipitation is not necessarily accompanied by denaturation and vica versa strong acids and bases can permanently destroy the H-bonding/salt-bridging network of proteins, denature and/or precipitate them; this is used in the lab to test for protein content (TCA, sulfosalicylic acid) ethanol or acetone can also precipitate proteins by shifting the dielectric constant of solvent water that results in lower solubility of solute protein heat denaturation/precipitation is of pathological relevance (high fever) Folding/refolding of proteins intriguing field of research for folding pathways refolding techniques are used and optimized to increase protein yield in heterologous protein expression and purification experiments (overexpressed excess protein may precipitate in the form of inclusion bodies that contain protein in a (partially) denatured insoluble form) refolding is not always spontaneous after dialysis of denaturant, helper materials are used to facilitate/initiate the folding process (native prosthetic groups/cofactors/substrates/ligands and e.g. PEG, arginine, CHAPS, lauril maltoside, glycerol, Triton X-100, BSA, etc. are good helper materials) a redox-shuffling system (Cys-cystine, GSH-GSSG, b-SH-EtOH, DTT) helps resolve wrongly made S-S bonds and find the thermodynamically most favorable conformation half-folded ?? sharp transition from the folded to the unfolded state (“all or none” cooperative process; same trend when the protein is refolded) there are transient intermediates of folding at the atomic level (progressive stabilization of intermediates); proteins may also get transiently stabilized in a molten globule form that contains native-like secondary structural elements but a rather dynamic tertiary structure somewhere in between the denatured and the native states) if one part of the protein structure is deteriorated (getting thermodynamically unstable under the given conditions), the whole structure will brake down (cooperatively) since the interactions that stabilized the rest of the protein are lost with this (unfolded) part of the enzyme What is the pathway to fold up? unfolded protein ? unique conformation in folded state the protein should try out all the possible conformations to find the energetically most favorable one? this would take for a 100 aa protein that samples 3 conformations/aa, each in 100 fs, ~1027 years (Levinthal`s paradox)……not a good option! Richard Dawkins in “The blind watchmaker” asked how long it would take for a monkey to spell out accidentally on a typewriter Hamlet`s remark to Polonius “Methinks it is like a weasel“…calculated…it would happen (probably) in about 1040 random keystrokes however, if we preserve the correct keystrokes and let the monkey retype only the wrong punches, the whole process would only take couple of thousands of trials! (cumulative selection, partly correct intermediates are retained) it is the way for a protein to correctly fold in a reasonable time frame to follow an at least partly defined folding pathway with intermediates on the road to the folded form (nucleation-condensation model, energy surface funnel model with multiple possible pathways to the same final stable structure at the bottom of the funnel, deepening in the energy-funnel means fewer and fewer conformations accessible to be adopted) Proteins successfully refolded by us Ambrus A et al., Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase. Prep. Biochem. & Biotechnol., 2001, 31(1): 59-70 Ambrus A, et al., Oligomerization of nitrophorins. Anal. Biochem., 2006, 352 (2): 286-95 Ambrus A et al., Refolding of the human dihydrolipoamide dehydrogenase. Biochem. Eng. J., 2009, 45(2): 120-125 Chaperones intracellular proteins assist in folding/preventing misfolding or aggregation of biomacromolecules and help assemble complex macromolecular structures, these proteins are called chaperones or chaperonins and some of them are even called foldases or unfoldases some chaperones assist in correctly folding newly synthesized protein chains as a minority of protein structures would not be able to correctly fold all by themselves they also assist in disassembling/unfolding of macromolecular structures they help assemble already folded structures to higher level structures (e.g. oligomers) they sometimes need co-chaperons to fully exhibit the chaperon action they do not convey “steric information” to fold a protein per se, they rather prevent transformation to non-functional structures they use sometimes ATP as an energy source for doing their folding action cellular shock (e.g. heat shock) leads to higher propensity of protein aggregation and specialized proteins, so-called “heat-shock proteins (HSP)”, help avoid this aggregation; not all chaperones are HSPs HSPs express as a response to higher T or other cellular shocks important chaperons (found especially in the ER): calnexin, calreticulin, different HSPs, protein disulfide isomerase, peptidyl prolyl cis/trans isomerase Hsp60 (GroEL/GroES complex in E. coli, Group I chaperonin, GroES is a cochaperonin) is the best characterized large (~ 1 MDa) chaperone complex, also found in the mitochondrial matrix other HSPs: HSP70 (prevent apoptosis), HSP90, HSP100, etc. (the number means MW) the mechanism of action generally requires ATP hydrolysis and major conformational changes from the chaperon`s side to be able to encapsulate the unfolded protein to the chaperon`s “lumen” where it will start folding Protein misfolding and aggregation – pathological relevance some infectious neurological diseases were recently revealed to be transmitted by virus-sized protein particles such examples are bovine spongiform encephalopathy (mad cow disease) and the analogous disease in humans, the Creutzfeldt-Jakob disease (CJD) the agents causing these diseases are called prions for proving the hypothesis that diseases can be transmitted purely by proteins, Stanley Prusiner in 1997 was awarded the Nobel Prize in Physiology or Medicine such proteins are massive, resistant to most regular treatments, aggregated proteins formed from a regular cellular, mostly helical, protein in the brain, PrP (prion protein); PrPSC is insoluble and of heterogenous state evidences say that helical and b-turn protein content gets converted to b-strand conformations that link to other b-strands of similar nature and form extended b-sheets and eventually protein aggregates (amyloids) the infectious agent in prion diseases is an aggregated form of a protein amyloids are insoluble fibrous protein aggregates sharing specific structural traits; abnormal accumulation of amyloids in organs may lead to amyloidosis that plays role in various (neurodegenerative) diseases (CJD, Alzheimer`s, Parkinson`s, Huntington`s diseases, Atherosclerosis, Diabetes mellitus type II, etc.) PrPSC nucleus (tau protein) normal PrP pool aggregation Ab-protein the protein-only model for prion disease transmission the disease can be transferredAb from one organism another by transis derived from theto cellular amyloid precursor ferring the nucleus (mad cow disease in the 1990sproteases; in the UK, protein outbrake (APP) through specific it is amyloid plaques in the prone to form insoluble animalssmall were intestine fed with feed of infected animal origin)aggregates and its structure by solid-state NMR spectroscopy showed extended parallel b-sheet arrangements Covalent protein modifications alter function acetylation of NH2-terminus makes proteins more stable against degradation hydroxylation of Pro stabilizes collagen fibers (implication of scurvy) lack of g-carboxilation of Glu in prothrombin leads to hemorrhage in Vitamin K deficiency secreted or cell-surface proteins are often glucosylated on Asn for being more hydrophylic and able to interact with other proteins addition of fatty acids to the NH2-terminus or Cys makes the protein more hydrophobic phosphorilation(/dephosphorilation) of Ser,Thr and Tyr alters the activities /function of enzymes (triggered generally by hormon or growth factor action on protein kinases/phosphatases, implications in signal transduction and regulation of metabolism) no new adduct, but a spontaneous rearrangement (and oxidation) of a tripeptide (Ser-Tyr-Gly) inside the protein occurs in green fluorescent protein (GFP, produced by certain jellyfish) that results in fluorescence (great tool as a marker in research) fluorescence micrograph of a 4-cell C.elegans embryo in which a PIE-1 protein labeled (covalently linked) with GFP is selectively emerges in only one of the cells (cells are outlined) some proteins are synthesized as inactive precursors (proprotein, zymogen) and stored until use; activation is possible via proteolytic cleavage (not to be mixed up with preproteins; preprotein=protein+signal peptide; many times first a pre-proprotein is synthesized that is cleaved then to the proprotein)