3 Å i i+1 i+2 CαCα CαCα CαCα The  (extended) conformation General shape

C’ top view side view N’ The  (extended) conformation Hydrogen bonds

The β conformation Some facts and statistics: 1.~30% of globular proteins 2.Strands lie side by side to form a sheet 3.Up to about10 residues per strand 4.3Ǻ rise per residue 5.Sidechains project upwards and downwards 6.Main-chain amides and carbonyls of different strands H-bond to reduce polarity

7.The sheet has a ~30º right twist 8.The sheet can be parallel, anti-parallel (β-meander) or mixed (only 20% of sheets) 9.Backbone amide-carbonyl H-bonds occur between strands Main-chain representation (β-meander)

The  (extended) conformation Structural motifs β-α-β motifTwisted β-sheet (thioredoxin)

The  (extended) conformation Structural motifs β-barrel

Why are helices and sheets so common? The energy change (in kcal/mol) of transferring a charged sphere from water into a hydrophobic environment: ΔE = E l – E w = 166 (q 2 /r) (1/ε l - 1/ε w )

c + Why are helices and sheets so common?

Why build helices and sheets? By pairing polar main-chain amide and carbonyl groups in H-bonds, helices/sheets electrostatically mask them from the hydrophobic core of the protein Disruption of one H-bond in the protein core: +5.3 kcal/mol (Ben-Tal et al, 1996) Disruption of 20 H-bonds (average helix): +106 kcal/mol Since the proteins are only marginally stable (5-20 kcal/mol), this means the disruption of protein structure!

Reverse turns and loops β-turns locations β-turn structure Loops

Antigen binding site including 6 loops Loops Connect secondary structure elements that create the hydrophobic core of the protein Usually hydrophilic and face the outside of the protein Hydrophilic nature results from polar residues and fewer satisfied main-chain H- bonds Often create binding/active sites of receptors and enzymes

Secondary elements are more ordered than loops

Tertiary Structure

Fibrous Globular Two types of proteins

Unfolded Folded Folded globular proteins have nonpolar core and overall polar surface

Globular proteins play different roles in numerous and diverse cellular activities (enzymes, transporters, immune, and regulatory proteins) This requires some properties that can only be conferred by the globular shape Globular proteins

The globular shape allows secondary structures to go in different directions This allows the protein to achieve: 1.Compactness (an advantage in the extremely dense cytoplasm) 2.Keeping hydrophilic residues outside (confers water solubility) while maximizing the burial of hydrophobic parts 3.Easy for creating binding sites (cavities) 4.Allows the joining of functional residues that are separated by sequence

Creation of binding site from residues separated by sequence

Some basic characteristics of tertiary structure Interactions that stabilize 3D structure: 1.Covalent interactions (disulfide) – less frequent because they limit protein dynamics 2.Non-covalent – vdW, electrostatic (ionic, H-bond), non-polar (hydrophobic) [reversible, confer specificity and allow dynamics] Aromatic ring stacking

The Ca 2+ -binding ‘EF-hand’ motif (Lewit-Bentely and Rety (2000)) Ca 2+ is involved in many signaling pathways in the cell, as well as in muscle contraction Ca 2+ works by binding to signaling proteins (e.g. calmodulin) and inducing conformational changes that allow further binding to other signaling proteins Helical motifs: helix-turn-helix

a b X, Y: Asp/Gln Z: Asp/Gln/Ser Y: Main-chain carbonyl X: H 2 O Z: Asp/Glu Helical motifs: helix-turn-helix EF-hand (Ca 2+ binding)

The Ca 2+ binding motifs in proteins are of limited configurations The most common motif is the ‘EF hand’, adopted also by bacteria This motif was first discovered in the muscle protein Parvalbumin (Krestinger) (Lewit-Bentley and Rety 2000) 1.1 Helix-loop-helix (HLH) motifs The motif is formed by the 5 th and 6 th helices (termed E, F) in parvalbumin, hence the name Based on this structure and the sequence constraints emerging from it, Krestinger predicted EF hand motifs in troponin C and calmodulin, which were later confirmed

(Lewit-Bentley and Rety 2000) The ‘hand’ analogy describes both the fold (helix-loop-helix) and the motion induced by Ca 2+ binding (a) The Ca 2+ -binding loop usually includes 12 residues with the pattern XYZG–Y– X–Z, where X, Y, Z, –X, –Y and –Z are the ligands that participate in metal coordination (b) and “” marks any amino acid In Parvalbumin, Ca 2+ is coordinated by the carboxylate sidechains of 5 residues (Asp/Glu), by main-chain carbonyl groups and by H 2 O The 6 th residue of the loop is Gly, preventing disturbance to the structure X, Y: ~ D/N Z: ~ D/N/S -Y:~peptide carbonyl -X:~ water -Z:~E/D

(Lewit-Bentley and Rety 2000) Some EF-hand motifs cannot bind Ca 2+ (e.g. p11). In these, the EF- hand conformation is maintained in the ‘open’ (analogous to Ca 2+ - loaded) form by a network of H-bonds (c) The motif is detected in small proteins (e.g. calmodulin), or within the domains of larger proteins (e.g. myosin or calpain) EF-hand motifs usually occur in pairs (two, or four in a dimer), with cooperative binding

c d Ca 2+ Free Bound myosin light chain Helical motifs: helix-turn-helix The EF-hand motif in calmodulin (CaM)

e f Helical motifs: helix-turn-helix The EF-hand motif in CaM

DNA-binding proteins (e.g. transcription factors) are able to recognize nucleotide sequences both specifically and non- specifically (the difference results from affinity) Helical motifs: helix-turn-helix

DNA TF Helical motifs: helix-turn-helix Helix-turn-helix (DNA binding)

Direct read-out of the DNA usually requires the protein to penetrate into the major and/or the minor grooves of the DNA One way to achieve this type of penetration is by using HTH motifs (the connection here is a short β-turn) The β-turn and first helix position the second helix in an orientation that allows it to fit inside the major groove of the DNA DNA-binding HTH motifs

DNA-binding HTH proteins are used by both bacteria and eukaryotes In eukaryotes, they serve in developmental regulation of gene expression Such are the ‘homeodomain proteins’, which contain an extended HTH motif DNA-binding HTH motifs

32 14  motifs β hairpin β meander Greek key

β-sheet  motifs The  sandwich motif

VLVL CLCL VHVH C H1 C H2 C H3 heavy chains light chains antigen binding site The immunoglobulin motif  motifs

Other  motifs  propeller  helix