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Proteins (8/17/07) unique chemistries specified by side chains (also backbone conformations!) Why do we have the L- form of AAs? R H R C NH3+ H C COO- COO- L isomer NH3+ D isomer mirror “Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity” Milton RC et al., Science. 1992 Jun 5;256(5062):1445-8 Proteins are polypeptides ..and so on… Implications……. C, O, N and H atoms of peptide bond are co-planar O O- N N+ 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group Trans vs. cis (non-prolines) 1000:1 Trans vs. cis (prolines) 3:1 D M Y G G H A Sequence (1º) S F L E A S Go here and look around!! Emil Fischer proposed a “lock and key” model for enzymes (1894) James B. Sumner was the first person to crystallize a protein (1926) Subsequent research has shown that “the folded” (native) state is actually an ensemble of highly similar structures The interior of proteins is hydrophobic Second Law of Thermodynamics “Elements in a closed system tend to seek their most probable distribution; in a closed system, Entropy always increases.” HYDROPHOBIC EFFECT Hydrophobic = “water fearing” Hydrophobia = “rabies” With the exception of integral membrane proteins, there is need for a way to put hydrophobic parts of polypeptide on inside and hydrophilic parts on outside! 2002 by W.H. Freeman and Company See direction? Famous Oregonian -helix 3.6 residues/turn 1.5Å rise/residue (also -, 310-helices) C=O of residue n h-bonds to NH of residue n+4 Pauling, Corey, and Branson predicted the -helix 6 years before it was seen in John Kendrew’s myoglobin structure. -helices involve h-bond from residue n to residue n + 5 (underwound) 310-helices have h-bonds from residue n to residue n + 3 (overwound) These types of helices occur rarely and are often found at the ends of -helices or as isolated single turns. Why? 2002 by W.H. Freeman and Company -helices are usually found along the outside of protein molecules with one side of the helix presenting hydrophobic residues to the interior, the other side presenting hydrophilic residues to the exterior (water). In globular proteins, helices pack by “ridges and grooves” Globin fold Separated by 3 Separated by 4 4 helix bundle 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group -sheets (built from different regions of polypeptide) parallel 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group Mixed (more rare) 3.5Å 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group antiparallel 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group Loops are on outside of molecule Loops have high content of charged and/or polar amino acids Loops connecting two antiparallel strands are called hairpin loops. Hairpin loops are usually short (3-6) but can be quite long (>20 residues). Except for very short loops (reverse turns), loops often have no regular structure. Copyright 1980 by W. H. Freeman and Company II I These have structure! C=O of i bonds To NH of i + 3 Do amino acids show preference for particular 2º structures? The RCSB Protein Structure Database (PDB) http://www.rcsb.org/pdb/ 45,213 structures (as of 8/14/07) Particular structures answer particular biological questions. The information afforded by ALL structures in the PDB can be applied toward general questions! What if one looked at the amino acid composition of various types of 2º structure? Chou and Fasman, Ann. Rev Biochem. 47 258 (1978), looked at the statistical distribution of amino acids in -helices, -sheet and turns and loops, using known protein structures from the protein databank. Much more recently… Protein Structure, Neighbor Effect, and a New Index of Amino Acid Dissimilarities .Xuhua Xia and Zheng Xie Mol. Biol. Evol. 19(1):58–67. 2002 Protein Structure, Neighbor Effect, and a New Index of Amino Acid Dissimilarities .Xuhua Xia and Zheng Xie Mol. Biol. Evol. 19(1):58–67. 2002 Note, for 2º prediction, context (neighbors) are also taken into consideration! Original Chou & Fasman Current protocols (with more structures and sequences) ~55% accurate ~80% accurate Cii Ci C’i-1 Ni+1 Ni Cii = 45º Ni = 45º Ci Cii C’i-1 = 180º Ni Ci Ni+1 Ni = -90º Cii = -90º Ci = 180º Copyright 1980 by W. H. Freeman and Company N N N N N C C = 0º C C C = 180º N ( = C—N—C—C) C = 90º Real Protein (High resolution structure) All non-glycine residues lie in most favored (red) and allowed (brown) regions Real Protein (Low resolution structure) Some non-glycine residues occupy the generously allowed (yellow) and disallowed regions (pale yellow) of the Ramachandran diagram Amino acid side chains adopt low energy configurations (rotomers) Introduction to Protein Structure (2nd edition),Branden&Tooze The primary sequence seems to be enough, in many cases, to guide folding. But how do proteins fold? 1969 Levinthal’s paradox – Can’t be by a random search of conformation space. Proteins fold by progressive stabilization of (correct) intermediates - not by a random search!! Structural Motifs We know that certain AA sequences favor -helices or -strands (or loops). Very often, AAs that are close in 1º structure end up close to each other in final folded structure. Adjacent 2ndry structural elements very often form structural “motifs” by packing against each other. C N DNA-binding N -hairpin EF hand Many proteins Helix-loop-helix Motifs are not typically independently stable. Domains, which are comprised (usually) of several motifs, are independently stable. C Motifs continued Helix above the plane -- Greek keys 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group Copyright 1980 by W. H. Freeman and Company Copyright 1980 by W. H. Freeman and Company “supersecondary” structure Coiled coil 140Å 2002 by W.H. Freeman and Company ionic g c a d f b 3.5 residues per turn Perfect repeat every 7 residues a e ionic g So-called “knobs in holes” packing 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group Collagen triple helix Gly-X-Y repeats parallel “-helices” 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group G-G-X-G-X-D loops 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group There’s even a four parallel sheet -helix from M. tuberculosis! Domains Are usually independently stable folds that very often contain recognizable structural motifs. Some whole proteins are a single domain. Many proteins are “mosaics” that are composed of many domains. What does this remind you of? This is why Jane Richardson said the first structure (myoglobin) was ‘…complex, irregular, and even ugly.’ Tertiary structure refers to the fold of a single polypeptide If the single polypeptide binds to another of its kind, the two form a homodimer and we’re now talking about quaternary structure. Many proteins exist as homo-multimers and many exist as hetero-multimers Glycerol kinase hemoglobin All structures The globin fold is seen in many disparate proteins and organisms. Homologies range from very high to quite low while still preserving the fold. / structures no regular motif Helix above the plane Active site crevice 1999 GARLAND PUBLISHING INC. A member of the Taylor & Francis Group Rossman fold (Lactate dehydrogenase) structures + Ubiquitin Tiam1 DH/PH Paste your sequence here A “mosaic” protein Click on domain Go to full annotation Here’s the sequence of the IQGAP1 GAP domain (992-1345) 341 of these! Here’s what a RasGAP domain looks like! What you should know One and three letter codes for amino acids Which amino acids are hydrophobic and which are charged or polar General features about helices, strands, and loops Phi/Psi angles Primary, secondary, tertiary, quaternary structure Maybe draw a topology diagram? Maybe predict secondary structure?