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Structure formation and association of biomolecules Prof. Dr. Martin Zacharias Lehrstuhl für Molekulardynamik (T38) Technische Universität München Motivation • Many biomolecules are chemically synthesized in a cell as linear chain molecules. • Chain molecules (proteins, RNA) need to form a defined three-dimensional structure: – Structure formation or folding process • The folded three-dimensional structure of a biomolecule is directly related to its function. Biomolecule function depends on threedimensional biomolecular structure. Protein-peptide complex Protein folding • Proteins can fold from an extended chain into a compact globular structure (some proteins need help to adopt the correct structure in vivo). • The fold is determined by the amino acid sequence. • The final protein three-dimensional (3D) structure has a well packed core. • The 3D-structure has limited stability • Folded structure can be disrupted by heat and changes of solution conditions Interactions of biomolecules • Most biomolecules form functional complexes • Many functions are mediated by multiple-protein-protein interactions • What are the driving forces for such interactions? • Specific vs. non-specfic interactions • What happens during a binding process? • Can we predict which proteins interact and how? + Outline • Protein structure – Folded and unfolded state • Molecular interactions and forces in solution • Thermodynamics of protein folding • Experimental methods to study protein folding • Theories of protein folding • Structure formation of nucleic acids • Computer simulation of structure formation • Mechanism of biomolecular association • Prediction of binding (geometry and affinity) • Talks on interesting papers Aim • Qualitative and quantitative understanding of folding and binding processes – What can be observed? – What are the current theories on folding and association • What are the forces that drive structure formation and folding? • Protein folding problem • Folding of nucleic acids • Prediction of 3D structures • Predicion of how biomolecules interact • Computer simulation of folding and binding Overview on protein structure (folded state) • Primary structure – Sequence of the protein • Secondary structure – Regular local structural elements stabilized by hydrogen bonds • Tertiary structure – Three-dimensional fold of the protein • Quaternary structure: – Non-covalent assembly of several proteins to form a functional complex -Gly-Ala-Arg-Phe-ValG A R F V Primary structure of proteins - Amino acid sequence of a protein: primary protein structure. - 20 naturally amino acids occur in proteins. - Amino acids have a common chemical structure: Tetrahedral (sp3) carbon atom (Cα) bound to four asymmetric groups: 1. Amino group (NH2) 2. Carboxy group (COOH) 3. H atom 4. Functional chemical group: characteristic for the amino acid - Naturally occuring amino acids are typically L amino acids. Cβ Cα NH2 Hα COOH The geometry of the peptide bond •Amino acids connect via a peptide bond -> poly-peptide structure. •Peptide bond formation by a condensation reaction •Peptide unit NH-CO (omega: ω) is planar (small deviations are possible) and is mostly in a trans configuration (except for Pro). R1 O phi N H O H psi O N omega R2 Amino acids can be grouped based on side chain character Non-polar side-chains (hydrophobic): Alanine (Ala), Valine (Val), Isoleucine (Ile), Leucin (Leu), Methionine (Met), Phenylalanine (Phe), Proline (Pro) and Tryptophan (Trp), also Glycine (Gly) Polar side-chains (hydrophilic): Serine (Ser), Threonine (Thr), Cysteine (Cys), Asparagine (Asn), Glutamine (Gln), Tyrosine (Tyr) and Histidine (His) Charged side-chains (hydrophilic): Positive charge: Arginine (Arg) and Lysine (Lys) Depending on the pH, Histidine (His) can also be positively charged. Negative charge: Glutamic acid (Glu) and Aspartic acid (Asp) Secondary structure of proteins • Regular structural elements which are formed by hydrogen bonds between relatively small segments of the protein sequence. • α-helix : – hydrogen bonds between the CO of residues i and the NH of residues i+4. – 3.6 residues per helical turn. – helical rise of 1.5 Å per residue. • Important secondary structures: – α-helix – β-sheet – turns – coil structures β-sheet + β-turn Super-secondary structures • • • To some degree tertiary structure can be viewed as a 3D 'packing' of secondary structural elements. Analysis of 3D protein structures indicates the existence of recurring patterns of secondary structure arrangements-> called motifs or super-secondary structure. common super-secondary structures (motifs) are: – β-hairpin – β-meander – βαβ-motif – Helix-loop-helix motif Examples: β-hairpin βαβ-motif helix-loop-helix motif Tertiary structure of proteins • The tertiary structure of a protein are the 3D coordinates to which it is folded. This tertiary structure is directly related to its function. • Some combinations of super-secondary elements are frequently found in proteins: – Four α-helix bundle (two α-helix-loop-α-helix motifs connected by a loop) – βαβαβ (Rossmann) fold (two βαβ motifs sharing one β-strands) • Not all helices and strands in a folded protein belong to supersecondary structural elements • Data base of protein structures: Brookhaven Protein Structure data base http://www.rcsb.org/pdb Classification of protein tertiary structures • Tertiary structure describes the packing of structural elements within one domain. • classification: – All (or mostly) α-helical: also called all-α – All (or mostly) β-sheet containing: all-β – α/β proteins: contain both helices and β-sheets – (sometimes additional class, α + β: segregated α-helices + β-sheets) – proteins with very little secondary structure content may form another class Spatial distribution of amino acids in folded proteins • The spatial distribution of amino acids with respect to the center of a folded globular protein is not random. • Hydrophobic amino acids are found preferentially inside the folded protein. • Hydrophilic and charged amino acids are more frequently located at the surface of the protein. • This observation indicates that the solvent plays a dominant role for the structure formation of a protein. • 3D-fold can be further stabilized by disulfide bonds. Protein domains • Domains: contiguous portions of the protein chain that fold into compact (semi-independent) tertiary structures (definition by Richardson, 1981). • Some proteins consists of only one domain. However, especialy proteins with several hundred residues often consists of several domains. • In some cases different functions of one protein can be associated with different domains. SHP2: Phosphotyrosine phosphatase 2 N-SH2-domain PTP-domain C-SH2-domain Is the number of possible 3D protein folds limited? • Observation – Many sequences form similar structures Atomic resolution structures of biomolecules are stored at the Protein Data Bank • • Contains ~35000 structures (mostly determined by X-ray crystallography) Several new structures per day Methods to determine the threedimensional structure of proteins • X-ray crystallography is the most powerful and most successful technique to obtain high resolution structures of a biomolecule – ~80% of all structures in the PDB have been solved by X-ray crystallography (X-ray diffraction of protein/nucleic acid crystals) – ~20% have been solved by NMR spectroscopy – Very few by other experimental techniques • A wide range of biomolecules and complexes can be analyzed by X-ray crystallography • X-ray crystallography requires high-quality (well ordered) single crystals of the biomolecule • NMR spectroscopy allows to determine the structure of a (small) protein in solution Examples of structures solved by X-ray crystallography nucleosome lysozyme K+ channel RNA-polymerase • Structures between a few hundred to up to million daltons can be solved if high-quality crystals can be obtained. ribosome The unfolded state of proteins • The term unfolded state can have several different meanings – Random coil structure of a peptide with negligible residual interactions (except chain connectivity) – The state formed immediately after synthesis (extended chain) – The state(s) formed after “denaturation” of a protein • Denaturation can be achieved by changing the temperature or the solution conditions. • What is the unfolded state of a protein? Random walk or stochastic chain model • Random walk or stochastic chains – Freely joined chain with constant bond length and no restriction of orientation of segments • The average (root mean square) end-to-end distance R for the chain scales with N0.5. • Very common (but also most unrealistic) model of an unfolded protein chain • • Experiment: Rg = Ro Nν, ν= 0.598+/-0.028 theoretical prediction for random coil according to rotational isomeric state model (Flory, 1969) for Θ-solvent: ν= 0.588 • Rigid segment simulations (connected by flexible linkers) • Rg = Ro Nν, ν= 0.602+/-0.028 (RIS for Θ-solvent: ν= 0.588) Energetic evaluation with a continuum solvent model Contributions to the total energy of a molecule conformation: Esolv-nonpolar + Esolv-polar ~surface area Poisson equation + + Force field + + + + Esolv-salt Poisson-Boltzmann eq + + + Evacuum - - + + + ΔGsolv= ΔGsolv(elec)+ΔGsolv(nonpolar) ΔGsolv(elec)= EPB(εin=2; εex=80)-EPB(εin=2; εex=1) ΔGsolv(nonpolar)= b + γ * SASA b = 0.86 kcal mol-1 γ = 0.005 kcal mol-1Å-2 Good agreement with experiment for PARSE set of optimized charges and radii „pure“ SASA-model Authors use two different solvation models to calculate σ and s for a zipper-model of (polyAla) α-helix folding All models give good agreement of calculated solvation free energy with experiment for a model compound (N-Methylacetamid): dGsolv(exp) = -10.0 kcal mol-1 Plotting ΔGi vs. i gives RTln(s) and RTln(s) as intercepts and slope, respectively. Improved continuum modelling of non-polar (hydrophobic) solvation • Continuum modelling of hydrophobic contribution using a single surface tension parameter: good predictions for solvation of linear alkanes, poor results for cyclic alkanes • Separation into two parts: A.) water reordering at molecule-water interface: (proportional to surface area) B.) molecule-water van der Waals interaction (surface integral) • • Significant improvement for cyclic alkanes Significant improvement for conformational changes Zacharias, M., J.Phys.Chem. 2003 cyclo-alkane branched+ n-alkanes