Introduction to the Structure of Macromolecules PDF

I nt ro d u c t i o n to t h e st r u c t u re o f m a c ro m o l e c u l e s Structure visualization Wire Stick Ball and stick ❑ Bonds-based representation ▪ Fast, little resource-demanding ▪ Suitable for detailed analysis ▪ Incorr...

I nt ro d u c t i o n to t h e st r u c t u re o f m a c ro m o l e c u l e s Structure visualization Wire Stick Ball and stick ❑ Bonds-based representation ▪ Fast, little resource-demanding ▪ Suitable for detailed analysis ▪ Incorrect impression about atom packing (empty space) and interatomic distances ❑ Hydrogen atoms are often omitted for simplicity Structure visualization 21 Structure visualization Helices Strands Loops Etc. Ribbon Cartoon ❑ Backbone-based representation ▪ Moderately fast, not very resource-demanding ▪ Suitable to investigate secondary structure and protein folds ▪ Shows main landmarks; good for overall orientation in the structure Structure visualization 22 Structure visualization CPK/ spheres Surface ❑ Surface-based representation ▪ Very slow, very resource-demanding ▪ Suitable to study shapes, volume, cavities and molecular contacts Structure visualization 23 Energetics of structures ❑ Energy ▪ Internal energy U (const. V); enthalpy H (constant P), … ▪ Total energy often inaccessible -> differences in energy ▪ Convention: negative energy is favorable, positive is unfavorable ▪ Potential energy Ep – interactions of atoms in a system ▪ Kinetic energy Ek – movement of atoms U = E p + Ek H = U + P.V Energetics of structures 25 Energetics of structures ❑ Entropy ▪ Related to the thermal disorder or conformational availability (degrees of freedom) ▪ Total entropy S > 0 ▪ Higher entropy is more favorable Energetics of structures 26 Energetics of structures ❑ Free energy ▪ Helmholtz A or F (const. V), Gibbs G (const. P) ▪ Combination of internal energy or enthalpy and entropy S A = U – TS ; G = H – TS → G = H - TS (T = temperature) ▪ Negative change of free energy (ΔG < 0) is favorable H↓ S↑ H↑ S↓ Energetics of structures 27 Energy landscape ❑ Relationship between structure and its potential energy ▪ Structure dictates potential energy – how strong are the individual interactions ▪ Potential energy reflects probability of finding the different structures – lower energy ➔ more frequently occurrence Transition ❑ Potential/free energy surface states ▪ Minima – stable structures ▪ Saddle points – transient ▪ Maxima – unstable structures State 1 Intermediate State 2 ▪ Energy barriers Energetics of structures 28 Energy landscape ❑ Relationship between structure and its potential energy ▪ Structure dictates potential energy – how strong are the individual interactions ▪ Potential energy reflects probability of finding the different structures – lower energy ➔ more frequently occurrence ❑ Potential/free energy surface ▪ Minima – stable structures ▪ Saddle points – transient ▪ Maxima – unstable structures ▪ Multidimensional surface Energetics of structures 29 Energy landscape ❑ Relationship between structure and its potential energy ▪ Structure dictates potential energy – how strong are the individual interactions ▪ Potential energy reflects probability of finding the different structures – lower energy ➔ more frequently occurrence Local maximum Saddle point Global maximum ❑ Potential/free energy surface ▪ Minima – stable structures ▪ Saddle points – transient ▪ Maxima – unstable structures Local minima ▪ Multidimensional surface Global minimum Energetics of structures 30 Molecular interactions ❑ Covalent interactions (chemical bonds) ▪ Between two atoms sharing electrons ▪ Very stable under standard condition ❑ Non-covalent interactions ▪ Much weaker than covalent bonds ▪ Electrostatic interactions ▪ Polar interactions ▪ Non-polar interactions Molecular interactions 32 Electrostatic interactions ❑ Charge-charge or ionic interactions ▪ Coulomb’s law – between any two charges ▪ Attractive (opposite signs) or repulsive (same sign) ▪ Long-range interactions (up to 10 Å) – decrease with r2 q1  q2 F= 4    r 2 r = distance  = permittivity Molecular interactions – electrostatics 33 Electrostatic interactions ❑ Charge-charge or ionic interactions q1  q2 F= ▪ Environment-dependent 4    r 2 ▪ Permittivity ε = ε0·εr ε0 = vacuum permittivity ▪ Relative permittivity (εr) = dielectric constant Non-polar Stronger force Highly polar Weaker force Molecular interactions – electrostatics 34 Electrostatic interactions ❑ Charge-charge or ionic interactions ▪ Environment dependent ▪ Salt concentration – presence of counter-ions (Na+, K+, Cl-, etc.) ▪ pH – may induce a change of charge Low pH (8) Very high pH (>10) Molecular interactions – electrostatics 35 Polar interactions acceptor (-) donor (+) ❑ Hydrogen bonds (H-bonds) ▪ Only between highly electronegative atoms: fluorine, oxygen, nitrogen (F, O, N) ▪ Donor and acceptor atoms sharing hydrogen ▪ H-bond distance: 2.8 – 3.4 Å  orbitals ❑ Aromatic (π-π) interactions ▪ Attractive interaction between aromatic rings ▪ Distance between the center of mass of rings: ~ 5 Å parallel displaced T-shaped sandwich Molecular interactions – polar 36 Polar interactions ❑ Van der Waals (vdW) interactions ▪ Between any two atoms ▪ Permanent dipole-dipole (in polar molecules) Molecular interactions – polar 37 Non-polar interactions ❑ Van der Waals (vdW) interactions ▪ Between any two atoms ▪ London dispersion forces, or temporary dipole-induced dipole (in non-polar molecules) ▪ Short-range interactions – up to 5 Å R1, R2 – van der Waals radii r - distance Molecular interactions – non-polar 38 Non-polar interactions ❑ Hydrophobic interactions ▪ Entropic origin – water molecules ordered around hydrophobic moiety -> unfavorable ▪ Hydrophobic packing -> favorable release of some ordered water molecules Molecular interactions – non-polar 39 Structure determination ❑ Established methods ▪ X-ray crystallography ▪ NMR spectroscopy ▪ Electron microscopy ▪ Bioinformatics predictions – theoretical Structure determination 42 Parameters of an X-ray structure ❑ Resolution ▪ Measure of the level of detail present in the diffraction pattern 3Å 2Å 1Å (bad) (acceptable) (exceptional) ❑ R-factor (residual factor; R-value) ▪ Measure of a model quality – i.e. the agreement between the crystallographic model and the diffraction data ▪ Varies from 0 (ideal) to 0.63 (random structure), typically about 0.2 Structure determination – X-ray crystallography 47 Parameters of an X-ray structure ❑ B-factors (thermal factors) ▪ Measure of how much an atom oscillates or vibrates around the position specified in the model ▪ Considered a measure of flexibility Structure determination – X-ray crystallography 48 X-ray crystallography ❑ Advantages ▪ No limitations in size ▪ Possibility to obtain an atomic resolution ❑ Disadvantages ▪ Requirement of a crystal ▪ Structure in a crystalline state (non-native) ▪ Static picture of macromolecule ▪ Position of hydrogen atoms (usually) are not detected Structure determination – X-ray crystallography 49 Parameters of an NMR structure ❑ RMSD ▪ Root-mean-squared deviation of atomic positions across the ensemble of solutions ▪ Reveals the mean differences between individual conformations ▪ Important parameter to compare different structures of the same molecule  = atom displacement ▪ ` N = total No. atoms Structure determination – NMR spectroscopy 52 NMR spectroscopy ❑ Advantages ▪ Structure in solution state (native) ▪ Possibility to investigate dynamics of macromolecules ▪ Position of hydrogen atoms detected ❑ Disadvantages ▪ Size limited to approximately 40 kDa (~ 400 amino acid proteins) ▪ Requirement of isotopically labeled sample Structure determination – NMR spectroscopy 53 Electron microscopy ❑ Advantages ▪ Applicable to extremely large systems ▪ Complements other methods e. g. X-ray, NMR ❑ Disadvantages ▪ Lower resolution (2-3 Å at best) Structure determination – electron microscopy 56 Bioinformatics predictions ❑ Homology modeling ❑ Machine learning ❑ Ab initio prediction Structure determination – bioinformatics predictions 57 Bioinformatics predictions Comparative modelling Ab initio predictions Amino acid sequence Amino acid sequence Find similar Homology search proteins in Profile method 3D structure Threading databases Machine learning Energy minimization, Molecular dynamics 3D structure database Predicted Predicted structure structure Structure determination – bioinformatics predictions 58 Bioinformatics predictions ❑ Homology modeling Comparison of sequences in databases: Multiple sequence alignment (MSA) Structure determination – bioinformatics predictions 59 Bioinformatics predictions ❑ Ab initio prediction Structure determination – bioinformatics predictions 61 Bioinformatics predictions ❑ Advantages ▪ Very fast (except ab initio) ▪ Low cost ❑ Disadvantages ▪ Ab initio is very demanding ▪ Theoretical model – experimental validation is needed Structure determination – bioinformatics predictions 62 S t r u c t u re o f b i o m o l e c u l e s Hierarchy of protein structure Proteins – hierarchy of protein structure 4  Amino acids Side ❑ 20 L-amino acids (natural) chain Amino ❑ Side chains group Chiral Acid ▪ Charged, polar, hydrophobic centre group Amino acid backbone Side chain - - + + Proteins – basic building blocks 5 Primary structure ❑ Linear chain of amino acid residues MSLGAKPFGEKKFIEIKGRRMAYIDEGTGDPILFQHGNPTSSYLWRNIM N-terminus C-terminus ❑ Protein backbone ▪ From N-terminus to C-terminus ▪ Connected by covalent bonds condensation ❑ Peptide bond (amide bond) -H2O ▪ Partial double bond character → Planar geometry Proteins – primary structure 6 Geometry of protein backbone ❑ Conformation of the peptide chain ▪ Defined by Φ (phi) and Ψ (psi) dihedral angles N+1 O C ❑ Ramachandran plot (Φ, Ψ) → The majority of proteins follow this distribution R 180 N C-1 O-1 Ψ φ (phi) = dihedral angle {C-1 − N − Cα − C} -180 ψ (psi) = dihedral angle {N − Cα − C − N+1} -180 Φ 180 Proteins – primary structure 7 Geometry of protein backbone ❑ Conformation of the peptide chain ▪ Defined by Φ (phi) and Ψ (psi) dihedral angles N+1 O C ❑ Ramachandran plot (Φ, Ψ) → The majority of proteins follow this distribution R N C-1 O-1 φ (phi) = dihedral angle {C-1 − N − Cα − C} ψ (psi) = dihedral angle {N − Cα − C − N+1} Proteins – primary structure 8 Secondary structure ❑ Local three-dimensional structure of polypeptide chain ❑ Governed by hydrogen bonding between backbone atoms ❑ Types of structures Helices ▪ Helices Strands Regular patterns Loops ▪ β-Structures ▪ Loops and coils - Irregular patterns Proteins – secondary structure 9 Helices ❑ Types of helices ▪ 3.613 helix (α-helix) – most common ▪ 310 helix – less frequent, end of α-helices ▪ 4.116 helix (π-helix) (rare) Left-handed ▪ Left-handed helix (very rare) α-helix → Represented by helical 310-helix cartoons or cylinders Ψ ❑ Right-handed (mostly) π-helix α-helix ❑ Hydrogen bonding ▪ Within a single chain Φ Proteins – secondary structure 11 Helices H-bonds 310 helix  Helix  helix Proteins – secondary structure 12 β-structures ❑ Types of typical β-structures ▪ β-sheets ▪ β-turns ▪ β-bulge ▪ Polyproline helices polyproline helices β-sheets ❑ Hydrogen bonding Ψ ▪ Between adjacent chains Φ Proteins – secondary structure 13 β-structures ❑ Types of β-sheets ▪ Parallel ▪ Antiparallel (stronger) ▪ Mixed → Represented by ribbons H-bonds with arrows indicating the sequence direction ❑ Side-chains ▪ Towards the sides of the sheets Proteins – secondary structure 14 Tertiary structure ❑ Global three-dimensional structure of protein ❑ Governed mainly by hydrophobic interactions involving side chains of amino acid residues Proteins – tertiary structure 18 Tertiary structure ❑ Supersecondary structures (motifs) ▪ Small substructures formed by several secondary structures ❑ Domain ▪ Structurally (functionally) independent regions ▪ Compact parts of structure – around single hydrophobic core ▪ Formed in separate folding unit (fold independently) ❑ Fold ▪ General architecture of protein ▪ Type of protein structure Proteins – tertiary structure 19 Quaternary structure ❑ Association of several protein chains (monomers/subunits) into oligomers (multimers) ▪ Homomeric protein – from identical monomers ▪ Heteromeric protein – from different types of monomers Homotetramer Heterodimer Heterotetramer hemoglobin tryptophan synthase immunoglobulin Proteins – quaternary structure 27 Nucleic acids Nucleic acids 28  Structure of nucleic acids… 29 Nucleotides ❑ Composition Nucleotide Nitrogenous base ❑ Phosphate ❑ Pentose sugar ❑ Heterocyclic base charge Sugar ❑ DNA bases: A, T; G, C ❑ RNA bases: A, U; G, C ❑ Rotation about glycosidic bond The anti conformation is dominant in DNA with rare exceptions Nucleic acids – basic building blocks 30 Primary structure ❑ Linear chain of nucleotides (oligonucleotides or polynucleotides) CGCGAATTCGCG ❑ Sugar-phosphate backbone ▪ Covalent character ▪ Phosphodiester bond ▪ From 5’-end to 3’-end Nucleic acids – primary structure 31 Primary structure ❑ Linear chain of nucleotides (oligonucleotides or polynucleotides) CGCGAATTCGCG ❑ Sugar-phosphate backbone ▪ Covalent character ▪ Phosphodiester bond ▪ From 5’-end to 3’-end oligonucleotide dGCAT (d indicates deoxyribose sugar, or a DNA sequence) Nucleic acids – primary structure 32 Sugar-phosphate backbone ❑ Very flexible backbone ▪ Six torsion angles ❑ Ribose is not planar → sugar puckering ▪ Denotes the phosphate-phosphate proximity ▪ Two main types of conformation 2′-deoxyribose (in DNA) Nucleic acids – primary structure 33 Secondary structure ❑ Local interactions between nucleotide bases →Base pairs ❑ DNA base pairs: Adenine - Thymine Cytosine - Guanine H-bonds ❑ RNA base pairs: Adenine - Uracil Cytosine - Guanine ❑ Complementarity due to hydrogen bonds Nucleic acids – secondary structure 34 Tertiary structure of DNA ❑ Overall three-dimensional arrangement and folding ❑ Three types: A-DNA, B-DNA, Z-DNA ❑ B-DNA is the most common (described by Watson & Crick) A-DNA B-DNA (rare) (predominant!) Z-DNA (rarer) Type A-DNA B-DNA Z-DNA Helix sense Right Right Left Bases per turn 11 10.5 12 Helical rise per nucleotide (Å) 2.6 3.4 3.7 C2’-endo Sugar pucker C3’-endo C2’-endo C3’-endo Nucleic acids – tertiary structure of DNA 36 Tertiary structure of DNA ❑ Grooves: crucial for DNA-protein interactions ❑ Major groove: wide and deep – where most proteins interact ❑ Minor groove: narrower and shallower Major 22 Å Groove 360 ~ 10 base pairs 34 Å Minor 12 Å Groove Sugar-phosphate backbone Nucleic acids – tertiary structure of DNA