Visualizing proteins with PyMol

Size: px

Start display at page:

Download "Visualizing proteins with PyMol"

Warren Sutton
5 years ago
Views:

Visualizing proteins with PyMol Structural Bioinformatics GENOME 541 Spring 2018 Lecture 1: Protein Structure Frank DiMaio (dimaio@uw.

1 Visualizing proteins with PyMol Structural Bioinformatics GENOME 541 Spring 2018 Lecture 1: Protein Structure Frank DiMaio Obtaining PyMOL We will use an educational build of PyMOL that is freely available. I will you download instructions this afternoon. Very Basic Tutorial Here is a simple tutorial that will show you how to open and visualize a protein: For today s lecture I will be using PyMOL to look at the protein ubiquitin. (pdb file: 1ubq from the Protein Data Bank - (pymol: show how to move protein, explain default representation) Motivation: Why do we care about macromolecular structure? Sequence --> Structure --> Function Structure determines function, so understanding structure helps our understanding of function Structure more conserved than sequence Structure allows identification of more distant evolutionary relationships Structure is encoded in sequence Understanding the determinants of structure allows design and manipulation of proteins Protein structural bioinformatics problems Analysis of protein structural properties Structure prediction from sequence Function prediction from sequence and structure Modeling molecular motions Interfaces, active sites, regulatory sites, specificity Predicting physical properties (stability, binding affinities) Design of structure and function 1

2 Proteins are Polymers of Amino Acids Proteins are Polymers of Amino Acids Amino acids Amino acids Amino acids have chiral centers polypeptide Water and hydrogen bonds Hydrogen bonds in general NOTE: H-bond distances vary. The H-bond distance shown is only the approximate average H-bond distance in liquid water. (see AAM, Table 2.3) O-O distance: nm = 2.74 Å = 1.77 Å = Å Lehninger 4/e Fig 2.1 Important: The O-H distance of ~2.74 Å in an H-bond is smaller than the sum of : (i) the O-H covalent bond distance of ~0.97 Å + (ii) the H vdw-radius of ~1.2 Å + (iii) the O vdw-radius of ~1.4 Å, since this sum is: = ~ 3.6 Å. 10 Å = 1 nm = 10-9 m In general: A hydrogen bond can be represented as D-H. A, where: D-H = weakly acidic donor group, such as O-H, N-H A = weakly basic acceptor atom such as O, N 2

3 Non-polar or Hydrophobic Amino Acids Glycine Glycine(Gly) (Gly, G) Alanine(Ala) (Ala, A) Valine Valine(Val) (Val, V) Isoleucine Isoleucine(Ile) (Ile, I) Leucine Leucine(Leu) (Leu, L) Polar or Hydrophilic Amino Acids OH Serine (Ser, S) Threonine (Thr, T) Cysteine (Cys, C) Asparagine (Asn, N) Glutamine (Gln, Q) Serine(Ser) Cysteine(Cys) Asparagine(Asn) Threonine(Thr) Glutamine (Gln) CH CH H CH 3 3 CH 3 CH CH 3 CH CH 3 OH CH CH 3 OH SH C O NH 2 C O CH 3 CH 3 NH 2 Phenylalanine(Phe) (Phe, F) Tyrosine(Tyr) (Tyr, Y) Trptophan(Trp) (Trp, W) Methionine Methionine(Met) (Met, M) Proline(Pro) Histidine(His) (Pro, P) (pk a =6.0) Proline(Pro) Aspartic Acid(Asp) Glutamic Acid(Glu) Lysine(Lys) Arginine(Arg) Histidine Histidine(His) (His, H) Aspartic Acid (Asp, D) Glutamic Acid (Glu, (pk (pk a=3.9) a=4.1) (pk E) Lysine a = 10.8) (Lys, K) Arginine (Arg, R) pka (pk = (pk a = 12.5) a=6.0) pka = 3.9 pka = 4.1 pka = 10.8 pka = 12.5 OH HN Serine(Ser) Cysteine(Cys) Asparagine(Asn) Glutamine (Gln) S CH 3 N H N NH O C Backbone bonds: red Side chain bonds: black N H N NH C O - O (pymol 1ubq show how to display sequence, explain atom coloring, select a specific amino acid type) C O - O NH 3+ NH +C NH 2 NH 2 The Building Blocks of All Proteins A Polypeptide Chain Gly Ala Val Ser Ile Leu Met Cys Thr Asn His Lys Arg Asp Glu Phe Gln Tyr Trp Pro Linking amino acids by forming peptide units. The order of the amino acids is called the Primary Structure of a protein 3

General Features of Polypeptides Ramachandran (Φ,Ψ) Plot Backbone has two polar groups per

lengths are largely invariant, proteins adopt different conformations by varying phi and psi

Ramachandran angles (using an alanine tripeptide) Sidechain dependence of Ramachandran

4 General Features of Polypeptides Ramachandran (Φ,Ψ) Plot Backbone has two polar groups per residue peptide bonds have double bond character and prefer to be planar Bond angles and lengths are largely invariant, proteins adopt different conformations by varying phi and psi (pymol -> show how to measure distances, angles and torsions) Interactive example of Ramachandran angles (using an alanine tripeptide) Sidechain dependence of Ramachandran angles Torsion preferences vary for different sidechains Most look like alanine because of clashes with Cβ 4

Classifying protein structure Higher-order Structure 1. Their primary structure is the amino acid sequence of the polypeptide chain. 2.

Tertiary structure refers to the three-dimensional structure of the entire polypeptide chain. 4.

(pymol -> show cartoon representation) Protein Secondary Structure: The a-helix Amphipathic a-helix Purple: Hydrogen Bonds Yellow: hydrophobic amino acids

5 Classifying protein structure Higher-order Structure 1. Their primary structure is the amino acid sequence of the polypeptide chain. 2. Secondary structure is the local spatial arrangement of a polypeptide s backbone atoms. Common secondary structures are a-helices and b-strands. 3. Tertiary structure refers to the three-dimensional structure of the entire polypeptide chain. 4. Some proteins are composed of two or more polypeptide chains. The spatial arrangement of these chains is a protein s quaternary structure. (pymol -> show cartoon representation) Protein Secondary Structure: The a-helix Amphipathic a-helix Purple: Hydrogen Bonds Yellow: hydrophobic amino acids Blue: hydrophylic amino acids i+2 i+1 i i+3 Red: Oxygen Dark Blue: Nitrogen Light Blue: Hydrogen Green: Carbon Val Lys Glu Leu Leu Asp Lys Val - Glu 3 4 i+4 A standard a-helix has hydrogen bonds between residues i and i+4. ( pymol show hydrogen bonds in helix) 5

3/26/18 Parallel vs Antiparallel b-strand Interactions Protein Secondary

Nitrogen Light Blue: Hydrogen Green: Carbon b-sheet b-strands come

also many MIXED ß-sheets, with some strands parallel and others

0 Å Cβ of side chain Lehninger f04.07 4th ed.

6 3/26/18 Parallel vs Antiparallel b-strand Interactions Protein Secondary Structure: The b-strand Purple: Hydrogen Bonds Red: Oxygen Dark Blue: Nitrogen Light Blue: Hydrogen Green: Carbon b-sheet b-strands come together to form b-sheets (the interaction can be either parallel or anti-parallel). ( pymol show beta sheets) β-sheets form a pleated sheet Note: There are also many MIXED ß-sheets, with some strands parallel and others antiparallel Parallel b-strands: why are they twisted? 7.0 Å Cβ of side chain Lehninger f th ed. VVP 2/e Fig 6-10 In both parallel and anti-parallel β-sheets: The side chains point alternatingly in opposite directions A fully extended chain is flat 23 Real beta strands twist and are not flat 6

Parallel b-strands: why are they twisted?

right-handed twist to peptide backbone; favored by side chains larger than Ala For a given energy, more accessible states for

patterning in beta sheet) Protein Secondary Structure: Loops and Turns Example: an antigen binding domain of an antibody

loop Between secondary and tertiary structure Supersecondary structure: arrangement of elements of same or different

7 Parallel b-strands: why are they twisted? Hydrophobic / hydrophilic patterning in b-strands On diagonal: no twist to peptide backbone phi=-psi Above diagonal: right-handed twist to peptide backbone; favored by side chains larger than Ala For a given energy, more accessible states for right-hand twist than for left-hand Thr Leu Asn Ile Lys - Phe (pymol -> show twist in sheet) 2 (pymol -> show hydrophobic patterning in beta sheet) Protein Secondary Structure: Loops and Turns Example: an antigen binding domain of an antibody Active site residues and binding residues are often found in loops. loop Between secondary and tertiary structure Supersecondary structure: arrangement of elements of same or different secondary structure into motifs; a motif is usually not stable by itself. Domains: A domain is an independent unit, usually stable by itself; it can comprise the whole protein or a part of the protein. Turns are short loops (2-4 residues), and typically have more regular structure than loops. 7

3/26/18 b-hairpin: Most common form of tight turn type Fi+1 Yi+1 Fi+2 Yi+2

Most common form of tight turn i+3 i+2 Example of a b-hairpin in bovine

Example of a protein with two bhairpins: erabutoxin from whale.

motif is characteristic of proteins binding to the major DNA grove.

8 3/26/18 b-hairpin: Most common form of tight turn type Fi+1 Yi+1 Fi+2 Yi+2 I I II II b-hairpin: Most common form of tight turn i+3 i+2 Example of a b-hairpin in bovine pancreatic trypsin inhibitor BPTI. Example of a protein with two bhairpins: erabutoxin from whale. i+1 i Type II The helix-turn-helix motif The helix-turn-helix motif This motif is characteristic of proteins binding to the major DNA grove. The proteins containing this motif recognize palindromic DNA sequences. The second helix is responsible for nucleotide sequence recognition. 8

βαβ motif Triose Phosphate Isomerase (TIM) A domain which occurs in a many proteins.

Accessibility to helix termini for hydrogen bonding? Trapped ends?

dimensional structure that is essential to the biological role they perform.

9 βαβ motif Triose Phosphate Isomerase (TIM) A domain which occurs in a many proteins. 5 4 Note the β-barrel in the center surrounded by α-helices Why? Shorter connections in right-handed topology? Accessibility to helix termini for hydrogen bonding? Trapped ends? Note the 8-fold repeated β-α motif The TIM barrel : α/β class topology 34 Protein Tertiary Structure Most proteins adopt a unique three dimensional structure that is essential to the biological role they perform. Protein structures can be divided into three groups: globular proteins, fibrous proteins, and integral membrane proteins. Examples: Most globular proteins share these characteristics 1) Hydrophobics on the inside 2) Close packing 3) Most polar groups involved in a hydrogen bond HIV protease (globular) Porin (membrane) Collagen (fibrous) Hydrophobic residues of procarboxypeptidase 9

Most globular proteins share these characteristics 1) Hydrophobics on the inside 2) Close packing 3) Most polar

and a backbone carbonyl (pymol) Fibrous Proteins Collagen highly elongated molecules that generally function as

the major stress-bearing component of bone, tendon and other connective tissues.

Collagen Gly Hyp Pro Gly Hyp = 4-hydroxyproline 3000 Å Pro is converted to Hyp by the enzyme prolyl hydroxylase which

10 Most globular proteins share these characteristics 1) Hydrophobics on the inside 2) Close packing 3) Most polar groups involved in a hydrogen bond Most globular proteins share these characteristics 1) Hydrophobics on the inside 2) Close packing 3) Most polar groups involved in a hydrogen bond acylphosphatase Hydrogen bond between a serine and a backbone carbonyl (pymol) Fibrous Proteins Collagen highly elongated molecules that generally function as structural materials their sequences are usually highly repetitive Sequence: G-X-Y Pro Hyp 15 Å Example: Collagen is the major stress-bearing component of bone, tendon and other connective tissues. A fiber 1 mm in diameter can support 20 lbs. Collagen Gly Hyp Pro Gly Hyp = 4-hydroxyproline 3000 Å Pro is converted to Hyp by the enzyme prolyl hydroxylase which uses vitamin C as a cofactor hence vitamin C deficiency can lead to unstable collagen à connective tissue problems (aka scurvy) 10

FIBROUS PROTEINS: Keratin and coiled coil α-helices

proteins ~70% of therapeutics are directed towards

ion and solute transport 2) detection of external

Membrane Proteins: hydrophobic residues are found on the

hydrophilic sidechains yellow: hydrophobic sidechains

protein avoids placing main chain C=O and NH groups in

water Ribbon representation of porin protein Space

11 FIBROUS PROTEINS: Keratin and coiled coil α-helices α-keratin is the principal protein of mammalian hair, nails, skin. Membrane Proteins ~30% of human proteins are membrane proteins ~70% of therapeutics are directed towards membrane proteins Membrane proteins are important for: 1) ion and solute transport 2) detection of external signals, e.g. hormones 3) cell-to-cell recognition 41 Membrane Proteins: hydrophobic residues are found on the exterior Hydrophobic environment membrane blue: hydrophilic sidechains yellow: hydrophobic sidechains Membrane proteins are often either all-α or all-β The protein avoids placing main chain C=O and NH groups in the hydrophobic bilayer) Bacteriorhodopsin OmpF Porin water Ribbon representation of porin protein Space filling representation of porin protein LIPID BILAYER LIPID BILAYER α-helices crossing the membrane β-barrel crossing the membrane (pymol -> show 2POR) membrane 44 11

Multi-domain proteins Multi-domain proteins are very common Many proteins contain independent domains connected by linkers. It is common to combine recognition domains with activation domains.

The order of the symbols indicates the order of the domains SH3 SH2 Kinase Interesting fact: the human genome does not contain more types of protein domains than more primitive organisms, but rather

12 Multi-domain proteins Multi-domain proteins are very common Many proteins contain independent domains connected by linkers. It is common to combine recognition domains with activation domains. By piecing domains together in new ways it is possible to create new functions. Example: Src tyrosine kinase. The SH3 domain recognizes substrate and the kinase domain phosphorylates the substrate. The order of the symbols indicates the order of the domains SH3 SH2 Kinase Interesting fact: the human genome does not contain more types of protein domains than more primitive organisms, but rather just puts them together in more complicated ways. Domains are compact folded nodules of a protein chain Living organisms often string domains together into one protein chain and then modify each domain for a specific function 46 Intrinsically Unfolded Proteins What are unstructured proteins? Proteins or segments of proteins that lack a well-structured 3D fold. They are referred to as natively unfolded or intrinsically unfolded How prevalent are unstructured proteins? Approximately 40% of proteins have unstructured regions that are longer than 50 residues, 6-17% of proteins in Swiss-Prot are probably fully disordered (based on theoretical predictions). What are the functions of unstructured proteins? There are many (see later). Many Intrinsically Unfolded Proteins Adopt Structure Upon Binding Partner Molecules Dyson and Wright (2005) Nat Rev Mol Cell Biol. 6:

Pliable (unstructured) proteins can interact with many different binding partners. Chaperones often contain unstructured regions that are used to recognize a diverse array of substrates.

13 What are some of the unique features of disordered proteins? Extensive binding interfaces can be created with relatively small proteins Conformational flexibility allows a protein segment to bind its target as well as to a modifying enzyme (i.e. posttranslational modification). Pliable (unstructured) proteins can interact with many different binding partners. Chaperones often contain unstructured regions that are used to recognize a diverse array of substrates. Classifying Tertiary Structure Historically: Much work done by Chothia to develop rules governing packing arrangements of secondary structure (like ridge-into-groove model for helix-helix packing) Modern schemes use sequence similarity and structurestructure comparisons to organize the protein universe and elucidate structural and evolutionary relationships SCOP structural classification of proteins CATH class architecture topology homologous superfamily Dali domain dictionary CATH a combination of manual and automated hierarchical classification four major levels: Class (C) based on secondary structure content Architecture (A) based on gross orientation of secondary structures Topology (T) based on connections and numbers of secondary structures Homologous superfamily (H) based on structure/function evolutionary commonalities provides useful geometric information (e.g. architecture) partial automation may result in examples near fixed thresholds being assigned inaccurately SCOP a purely manual hierarchical classification three major levels: Family based on clear evolutionary relationship (pairwise residue identities between proteins are >30%) Superfamily based on probable evolutionary origin (low sequence identity but common structure/function features Fold based on major structural similarity (major secondary structures in same arrangement and topology provides detailed evolutionary information manual process influences update frequency and equally exhaustive examination 13

α-helical proteins β proteins Mostly β strands Many antiparallel strand pairs

arrangements β sandwich two sheets paired with ~30 rotation α/β proteins Other

that use disulfide bonds to confer stability coiled coils: 2+ parallel helices

βαβ elements: mostly parallel strand pairs Open faced sheets with two edges; two

14 α-helical proteins β proteins Mostly β strands Many antiparallel strand pairs from β hairpins Mostly α-helices Parallel/antiparellel bundles Globular arrangements β sandwich two sheets paired with ~30 rotation α/β proteins Other protein classes α+β: segregated α and β secondary structure small: tiny proteins that use disulfide bonds to confer stability coiled coils: 2+ parallel helices wrapped around one another Intermixed α and β secondary structure elements Many βαβ elements: mostly parallel strand pairs Open faced sheets with two edges; two or three layers Barrels with no edge strands Helices tend to follow the direction of strands 14

$perfect protein bombard with X-rays, record scattering diffraction patterns determine electron density$ map from scattering and phase via Fourier transform: use electron density and biochemical knowledge of

map from scattering and phase via Fourier transform: use electron density and biochemical knowledge of

aqueous solution, motile and tumbles/vibrates with thermal motion NMR detects chemical shifts of

between specific pairs of atoms based on shifts, constraints use constraints and biochemical knowledge

15 3/26/18 The protein structure universe X-Ray Crystallography crystallize and immobilize single, perfect protein bombard with X-rays, record scattering diffraction patterns determine electron density map from scattering and phase via Fourier transform: use electron density and biochemical knowledge of the protein to refine and determine a model NMR Spectroscopy determining constraints protein in aqueous solution, motile and tumbles/vibrates with thermal motion NMR detects chemical shifts of atomic nuclei with non-zero spin, shifts due to electronic environment nearby determine distances between specific pairs of atoms based on shifts, constraints use constraints and biochemical knowledge of the protein to determine an ensemble of models Cryo-electron microscopy using constraints to determine secondary structure 15

Structure formation and association of biomolecules. Prof. Dr. Martin Zacharias Lehrstuhl für Molekulardynamik (T38) Technische Universität München

Structure formation and association of biomolecules Prof. Dr. Martin Zacharias Lehrstuhl für Molekulardynamik (T38) Technische Universität München Motivation Many biomolecules are chemically synthesized