Introduction to Molecular Neurobiology Protein Structure Erez Podoly Introduction to Molecular Neurobiology

From Primary to Quanternary Structure 1º 2º 3º 4º 2º  helices and  sheets are stabilized by hydrogen bonds between backbone oxygen and hydrogen atoms. There are three major 2º components: helices, β-sheets and what’s in-between them (turns and loops).

The Protein Folding Problem In the 1960’s, C.B. Anfinsen performed a series of in vitro experiments that lead him to the “Thermodynamic Hypothesis”: As he stated ten years later, in his 1972 Nobel acceptance speech, “The native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment”. Anfinsen’s hypothesis has some exceptions: Some proteins have multiple conformations and some proteins get folding help from chaperones.

Chou and Fasman Favors -Helix Favors Favors turn Amino Acid -Helix β-Sheet Turn Ala 1.29 0.90 0.78 Cys 1.11 0.74 0.80 Leu 1.30 1.02 0.59 Met 1.47 0.97 0.39 Glu 1.44 0.75 1.00 Gln 1.27 0.80 0.97 His 1.22 1.08 0.69 Lys 1.23 0.77 0.96 Val 0.91 1.49 0.47 Ile 0.97 1.45 0.51 Phe 1.07 1.32 0.58 Tyr 0.72 1.25 1.05 Trp 0.99 1.14 0.75 Thr 0.82 1.21 1.03 Gly 0.56 0.92 1.64 Ser 0.82 0.95 1.33 Asp 1.04 0.72 1.41 Asn 0.90 0.76 1.23 Pro 0.52 0.64 1.91 Arg 0.96 0.99 0.88 Favors -Helix Favors β-strand Favors turn

Leventhal Paradox How much time does it take to a given protein (100aa) to fold into a single stable native conformation (assuming three conformations/peptide bond)?

Leventhal Paradox • 3100 = 5.15 × 1047 conformations. • Fastest motions 10-15 sec. • Sampling all conformations would take 5.15 × 1032 sec. • 60 × 60 × 24 × 365 = 3.15 × 107 seconds in a year. • Sampling all conformations will take 1.6 × 1025 years. • The age of the universe is ~ 11-20 × 109 years. The Leventhal Paradox: proteins are able to quickly fold into their conformations despite such an overwhelming number of possibilities.

Quick Overview of Energy Strength (kcal/mole) Bond 3-7 H-bonds 10 Ionic bonds 1-2 Hydrophobic interactions 1 Van der vaals interactions 51 Disulfide bridge

The Protein Folding Problem Proteins could fold more quickly if they retain native-like intermediates along the way.

The Protein Folding Problem Much of conformation space is already restricted by allowed phi/psi angles (Ramachandran plot). F Y Gopalasamudram Narayana Iyer Ramachandran (1922 – 2001)

The Dihedral Angles – Φ, Ψ Each unit can rotate around two such bonds: Cα-N (phi) & Cα-C (psi). Most combinations of Φ & Ψ angles are not allowed due to steric collisions. Note the Phi and Psi angles that determine the spatial position of the planes relative to each other. These are important determinants of secondary structure and will be discussed next week. Cα -N bond (phi) Cα -C bond (psi)

Ramachandran Plot The angle pairs Φ & Ψ are usually plotted against each other in a diagram called a Ramachandran plot. F Y Most of Ramachandran plot area includes values that are not allowed. Colored areas show sterically allowed regions.

Secondary Structures The major allowed regions in Ramachandran plot define conformational components of proteins.

Structural Classification of Proteins CLASS: , β, /β, +β (but also: multi-domain, membrane and cell surface, small proteins, coiled coil proteins). FOLD: secondary structures in same arrangement. SUPERFAMILY: function/structure similarity. FAMILY: >30% sequence similarity, and similar known structure/function. Protein Class Protein Fold Protein Superfamily Protein Family

Structural Classification of Proteins SCOP Manual classification http://scop.berkeley.edu/ CATH Semi-manual classification http://www.cathdb.info/latest/index.html FSSP Automatic classification http://ekhidna.biocenter.helsinki.fi/dali/start Class Folds Superfamilies Families All  218 376 608 All β 144 290 560 /β 136 222 629 +β 279 409 717 Multi-domain 46 61 Membrane & cell surface 47 88 99 Small proteins 75 108 171 Total 945 1539 2845

Proteins’ Classes

All  All β /β

Proteins’ Folds Proteins are defined as having a common fold if they have the same major secondary structures in the same arrangement and with the same topological connections. A structural domain is an element of overall structure that is self-stabilizing and often folds independently of the rest of the protein chain; Most domains can be classified into "folds". Because they are self-stabilizing, domains can be "swapped" by genetic engineering between one protein and another to make chimera proteins. http://pawsonlab.mshri.on.ca/index.php?option=com_content&task=view&id=30&Itemid=63

The Structure/function paradigm In parallel with the growth in structural knowledge, there has been an increasing conviction that the biological function of proteins is encoded in their 3D structure. Most molecular biologists believe that determining protein functions depends on the protein structure. The Structure/function paradigm: the amino acid sequence determines protein 3D structure and the structure determines the function.

Intrinsically Disordered Proteins A significant proportion of proteins contain regions, sometimes quite large, that apparently don't fold into specific structure, but rather remain as flexible ensembles. These regions are termed "intrinsically disordered”, but also “intrinsically unstructured” or “Naturally unfolded”. Disordered regions are sequences within proteins that fail to fold into one fixed structure. It doesn’t mean they don’t have a structure, on the contrary: we may consider them as “multiple folded”.

Expansion of the Structure/Function Paradigm “Creating a new theory is not like destroying an old barn and erecting a skyscraper in its place. It is rather like climbing a mountain, gaining new and wider views, discovering unexpected connections between our starting points and its rich environment. But the point from which we started out still exists and can be seen, although it appears smaller and forms a tiny part of our broad view gained by the mastery of the obstacles on our adventurous way up”. -- Albert Einstein, The Evolution of Physics. Function can arise from the two protein forms (the ordered state, but also the random coil state) and transitions between them. Thus, proteins that lack a 3D structure may carry out function.

The History of Structural Biology 1895: W. C. Roentgen discovers X rays. 1912: Max von Laue discovers X-ray diffraction by crystals. 1913: W. L. Bragg reports the crystal structure of NaCl . 1935: J. M. Robertson solves the structure of pthalocyanin. 1948: Bijvoet solves strychnine (cryst. decides bet. alternatives). 1958: Kendrew reports the crystal structure of Myoglobin. 1962: M. F. Perutz and Sir J. C. Kendrew win the Nobel Prize for their studies on the structures of globlular proteins. 1965: Lysozyme. 1968: Haemoglobin . 1971: Insulin. 1971: PDB is established at Brookhaven National Lab., NY.

Methods for Structure Determination

Methods for Structure Determination X-Ray NMR Rate limiting Step Crystallization Resonance assignment Physical principle Electron diffraction Spin of nuclei in magnetic field Hydrogens Invisible The only detectable atom # structures in solution 1 15-20 Size limitation None (MDa) 64KDa Wavelength Range 0.1 – 100 Å 0.6 – 10 m Quality measure Resolution and R factor RMSD The result Snapshot Protein movements Thermodynamics Not possible Kinetic measurements

Methods for Structure Determination NMR X-Ray