Week 3-3D structure of protein.pdf

THE THREE-DIMENSIONAL STRUCTURE OF PROTEINS Assist. Prof. Dr. Derya DİLEK KANÇAĞI Room Number: 511 E-mail: [email protected] Office Hour: Wednesday 13.00-15.00 Central Principles in Biochemistry Principle 1 • Protein structures are stabilized noncovalent interactions and forces. Principle...

THE THREE-DIMENSIONAL STRUCTURE OF PROTEINS Assist. Prof. Dr. Derya DİLEK KANÇAĞI Room Number: 511 E-mail: [email protected] Office Hour: Wednesday 13.00-15.00 Central Principles in Biochemistry Principle 1 • Protein structures are stabilized noncovalent interactions and forces. Principle 2 by • Formation of a thermodynamically favorable structure depends on the influences of the hydrophobic effect, hydrogen bonds, ionic interactions, and van der Waals forces. Natural protein structures are constrained by peptide bonds, whose configurations can be described by the dihedral angles φ and ψ. • Protein segments can adopt regular secondary structures such as the α helix and the β conformation. • These structures are defined by particular values of φ and ψ and their formation is impacted by the amino acid composition of the segment. All of the φ and ψ values for a given protein structure can be visualized using a Ramachandran plot. Central Principles in Biochemistry Principle 3 Principle 4 • Tertiary structure describes the welldefined, three-dimensional fold adopted by a protein. • Tertiary structure is determined by amino acid sequence. • Protein structures are often built by combinatorial use of common protein folds or motifs. Quaternary structure describes the interactions between components of a multisubunit assembly. Proteostasis is the process to maintain the homeostasis of the proteome, the building, and turnover of proteins. • Even though protein folding is complex, some denatured proteins can spontaneously refold into their active conformation based only on the chemical properties of their constituent amino acids. Cellular proteostasis involves numerous pathways that regulate the folding, unfolding, and degradation of proteins. Many human diseases arise from protein misfolding and defects in proteostasis. Central Principles in Biochemistry Principle 5 • The three-dimensional proteins can be defined. structures of • Structural biologists use a variety of instruments and computational methods to solve biomolecular structures. The choice of method may depend on factors such as the size of the protein being studied, its properties, or the desired resolution of the final structure. • In principle, proteins can assume an uncountable number of special arrangements, or conformations free rotation is possible • Chemical or structural functions relate to unique three-dimensional structures Relationship between protein structure and function Overview of Protein Structure The possible conformations of a protein or protein segment include any structural state it can achieve without breaking covalent bonds. Protein Conformations • Limited number of conformations predominate under biological conditions • Conformations = thermodynamically the most stable, that is, lowest free energy (G) • Native = proteins in any functional, folded conformations • In some cases, entire proteins are intrinsically disordered, yet are fully functional. A Protein’s Conformation Is Stabilized Largely by Weak Interactions • Stability = tendency of a protein to maintain a native conformation • Unfolded proteins have high conformational entropy • Chemical interactions stabilize native conformations: • Strong disulfide (covalent) bonds are uncommon • Weak (noncovalent) interactions and forces are numerous • Hydrogen bonds • Hydrophobic effect • Ionic interactions A given polypeptide chain can theoretically assume countless conformations Packing of Hydrophobic Amino Acids Away from Water Favors Protein Folding • Hydrophobic effect = predominating weak interaction • Solvation layer = highly structured shell of H2O around a hydrophobic molecule • Decreases when nonpolar groups cluster together • Decrease causes a favorable increase in net entropy • This increase in entropy is the major thermodynamic driving force for the association of hydrophobic groups in aqueous solution. • Hydrophobic R chains form a hydrophobic protein core Polar Groups Contribute Hydrogen Bonds and Ion Pairs to Protein Folding • Repeating secondary structures ( helices and  sheets) optimize hydrogen bonding • Interaction of oppositely charged groups = ion pair = salt bridge • Strength increases in an environment of lower dielectric constant, ε – polar aqueous solvent: ε ~ 80 either a stabilizing or – nonpolar protein interior: ε ~ 4 destabilizing effect on protein structure. The dielectric constant of a substance or material is a measure of its ability to store electrical energy. Salt bridges in proteins are bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction. Protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings - PubMed (nih.gov) Individual van der Waals Interactions Are Weak but Combine to Promote Folding • Van der Waals interactions = dipole-dipole interactions over short distances (0.3 nm-0.6 nm) • Individual interactions contribute little to overall protein stability • High number of interactions can be substantial • The number of hydrogen bonds and ionic interactions within the protein is maximized, thus reducing the number of unpaired hydrogen Bonding and ionic groups. The Peptide Bond Is Rigid and Planar • 3 covalent bonds separate the  carbons of adjacent amino acid residues: C —C—N—C • Resonance between the carbonyl oxygen and the amide nitrogen (partial sharing of two pairs of electrons) • Partial negative charge and partial positive charge sets up a small electric dipole Peptide C—N Bonds Cannot Rotate Freely • 6 atoms of the peptide group lie in a single plane • Partial double-bond character of C—N peptide bond prevents rotation, limiting range of conformations The planar peptide group Dihedral Angles Define Peptide Conformations • 3 dihedral angles: – φ (phi) = between −180 and +180 degrees – ψ (psi) = between −180 and +180 degrees – ω (omega) = ±180 degrees for trans The planar peptide group Prohibited Conformations • Many φ (phi) and ψ (psi) values are prohibited by steric interference • Φ and ψ cannot both = 0 degrees The planar peptide group Protein Secondary Structure Protein Secondary Structure • Secondary structure = describes the spatial arrangement of the main-chain atoms in a segment of a polypeptide chain • Regular secondary structure = φ and ψ remain the same throughout the segment • Common types = α helix, β conformation, β turn, random coils • Secondary structures without a regular pattern are sometimes referred to as undefined or as random coils. • The path of most of the polypeptide backbone in a typical protein is not random; rather, it is highly specific to the structure and function of that protein. The α Helix Is a Common Protein Secondary Structure • α helix = simplest arrangement, maximum number of hydrogen bonds • Backbone wound around an imaginary longitudinal axis • Every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid that is four residues earlier in the protein sequence. • R groups protrude out from the backbone • Each helical turn = 3.6 residues, ∼5.4 Å • Dihedral angles define protein conformations • Right-handed: • R groups protruding away from the helical backbone • Most common • Extended left-handed: theoretically less stable, not observed in proteins The angstrom, Å, named after the physicist Anders J. Ångström, is equal to 0.1 nm. Intrahelical Hydrogen Bonds • Between hydrogen atom attached to the electronegative nitrogen atom of residue n and the electronegative carbonyl oxygen atom of residue n + 4 • Confers significant stability Helical backbone is held together by hydrogen bonds between the backbone amides of an n and n + 4 amino acids. *Each successive turn of the helix is held to adjacent turns by three to four hydrogen bonds, conferring significant stability on the overall structure. At the ends of an α-helical segment, there are always three or four amide carbonyl or amino groups that cannot participate in this helical pattern of hydrogen bonding. These may be exposed to the surrounding solvent, where they hydrogen bond with water, or other parts of the protein may cap the helix to provide the needed hydrogen-bonding partners. Amino Acid Sequence Affects Stability of the α Helix • Not all polypeptides can form a stable α helix. • Amino acid residues have an intrinsic propensity to form an α helix • Interactions between R chains spaced 3–4 residues apart can stabilize or destabilize α helix • Charge, size, and shape of R chains can destabilize • Formation of ion pairs and hydrophobic effect can stabilize Alanine shows the greatest tendency to form αhelices in most experimental model systems. Helical wheel Proline and Glycine Occur Infrequently in an α Helix • Proline = introduces destabilizing kink in helix • Nitrogen atom is part of rigid ring • Rotation about N—Calfa bond not possible • Glycine = high conformational flexibility, take up coiled structures Amino Acid Residues Near the End of the α Helix Segment Affect Stability • Small electric dipoles in each peptide bond align through hydrogen bonds • Negatively charged amino acids often found near the NH3+ terminus (stabilizing effect) • Positively charged amino acids often found near the COO– terminus (destabilizing effect) Helix dipole. The β Conformation Organizes Polypeptide Chains into Sheets • β conformation = backbone extends into a zigzag • β strand = single protein segment • β sheet = several strands in β conformation side by side Adjacent Polypeptide Chains in a β Sheet Can Be Antiparallel or Parallel • Antiparallel = opposite orientation • Occur more frequently • Parallel = same orientation • H bonds form between backbone atoms of adjacent segments • Β turns are common in proteins • Β turns = connect ends of two adjacent segments of an antiparallel β sheet • 180° turn • Involves 4 residues • Hydrogen bond forms between first and fourth residue • Gly (residue 2) and pro (residue 3) often occur in β turns In natural proteins, antiparallel β sheets are found twice as frequently as parallel β sheets Structures of β turns Common Secondary Structures Have Characteristic Dihedral Angles • Dihedral angles φ (phi) and ψ (psi) associated with each residue completely described secondary structure • Ramachandran plots: • Visualize all φ and ψ angles • Test quality of three-dimensional protein structures • Secondary Structure Conformations are Defined by φ and ψ Values • φ and ψ Values from Known Proteins Fall into Expected Regions • Glycine frequently falls outside the expected ranges Common Secondary Structures Can Be Assessed by Circular Dichroism • Circular dichroism (CD) spectroscopy = measures differences in the molar absorption of lefthanded vs. right-handed circularly polarized light:  = L – R • For proteins, spectra are obtained in the far UV region (190 to 250 nm). In this region, the lightabsorbing entity, or chromophore, is the peptide bond; a signal is obtained when the peptide bond is in a folded environment. • Different Secondary Structures Have Different Circular Dichroism Spectra Circular dichroism spectroscopy (1) Circular Dichroism spectroscopy in 4 minutes - YouTube Protein Tertiary and Quaternary Structures Protein Tertiary and Quaternary Structure • Tertiary structure = overall three-dimensional arrangement of all the atoms in a protein • weak interactions and covalent bonds hold interacting segments in position • Quaternary structure = arrangement of 2+ separate polypeptide chains in three-dimensional complexes • Four major types of protein groups based on polypeptide chains: • Fibrous proteins = arranged in long strands or sheets • Globular proteins = folded into a spherical or globular shape • Membrane proteins = embedded in hydrophobic lipid membranes • Intrinsically disordered proteins = lacking stable tertiary structures • Fibrous proteins usually consist of a single type of secondary structure, and their tertiary structure is relatively simple. Globular proteins often contain several types of secondary structure. • Most enzymes are globular proteins, whereas regulatory proteins can be globular, disordered, or contain both globular and disordered segments. Fibrous Proteins Are Adapted for a Structural Function • Give strength and/or flexibility to structures • Simple repeating element of secondary structure • H2O insoluble due to high concentrations of hydrophobic residues Structural Diversity Reflects Functional Diversity in Globular Proteins • Globular proteins: • Fold back on each other • More compact than fibrous proteins • Enzymes, transport proteins, motor proteins, regulatory proteins, immunoglobulins • Globular Proteins Have a Variety of Tertiary Structures • Each globular protein has a distinct structure, adapted for its biological function The Protein Data Bank • The Protein Data Bank (PDB): www.rcsb.org • • • • Archive of experimentally determined three-dimensional structures Structures assigned an identifier called the PDB ID PDB data files describe: • the spatial coordinates of each atom • information on how the structure was determined • information on its accuracy Structure visualization software can convert atomic coordinates to an image of the molecule Folding Patterns of Proteins To understand a complete three-dimensional structure, we need to analyze its folding patterns • Motif = fold = recognizable folding pattern involving 2+ elements of secondary structures and the connection(s) • Can be simple, such as in a β-α-β loop • Can be elaborate, such as in a β barrel • Domain = part of a polypeptide chain that is independently stable or could undergo movements as a single entity • Domains may appear as distinct or be difficult to discern • Small proteins usually have only one domain • Different domains often have distinct functions, such as the binding of small molecules or interaction with other proteins. Small proteins usually have only one domain (the domain is the protein). Protein-Folding Rules • Burial of hydrophobic R groups requires 2+ layers of secondary structure • α helices and β sheets are found in different layers • Adjacent amino acid segments are usually stacked adjacent • The β conformation is most stable with right-handed connections Stable folding patterns in proteins Complex Motifs Are Built from Simple Motifs • α/β barrel = series of β-α-β loops arranged such that the β strands form a barrel Some Proteins or Protein Segments Are Intrinsically Disordered Binding of the intrinsically disordered carboxyl terminus of p53 protein to its binding partners • Intrinsically disordered proteins: • Lack definable structure • Often lack a hydrophobic core • High densities of charged residues (lys, arg, glu) and pro • Facilitates a protein to interact with multiple binding partners • Intrinsically Disordered Segments Can Assume Different Structures Although many proteins contain well-folded and stable structures, this is not necessary for the biological function of all proteins. The lack of an ordered structure can facilitate a kind of functional promiscuity, allowing one protein to interact with multiple or even dozens of partners. Protein Motifs Are the Basis for Protein Structural Classification • Protein Data Bank (PDB) = 150,000+ structures archived • Structural Classification of Proteins database (SCOP2) = searches protein information in the PDB • (1) protein relationships, (2) structural classes, (3) protein types, and (4) evolutionary events. The number of folding patterns is not infinite. Among the tens of thousands of distinct protein structures archived in the PDB, only about 1,400 different folds or motifs are classified by the SCOP2 database. Given the many years of progress in structural biology, new motifs are now discovered only rarely. Topology Diagrams • Topology diagram = represent elements of secondary structure and the relationships among segments of secondary structure in a protein Protein Families and Superfamilies • Proteins with significant similarity in primary structure and/or tertiary structure and function are in the same protein family • ~4,000 different protein families in the PDB • Strong evolutionary relationship within a family • Superfamilies = 2+ families that have little sequence similarity, but the same major structural motif and have functional similarities • Evolutionary relationship is probable Protein Quaternary Structures Range from Simple Dimers to Large Complexes • • Quaternary structure = assembly of multiple peptide subunits Oligomer = multimer = multisubunit protein • Repeating structural unit = protomer The first oligomeric protein to have its threedimensional structure determined was hemoglobin (Mr 64,500), which contains four polypeptide chains and four heme prosthetic groups, in which the iron atoms are in the ferrous (Fe2+) state. Protein Denaturation and Folding Pathways Involved in Proteostasis • Proteostasis = continual maintenance of the active set of cellular proteins required under a given set of conditions • First, proteins are synthesized on a ribosome. • Second, various pathways contribute to protein folding, many of which involve the activity of complexes called chaperones. Chaperones (including chaperonins) also contribute to the refolding of proteins that are partially and transiently unfolded. • Finally, proteins that are irreversibly unfolded are subject to sequestration and degradation by several additional pathways. Loss of Protein Structure Results in Loss of Function • Denaturation = loss of three-dimensional structure sufficient to cause loss of function • Can occur by heat, pH extremes, miscible organic solvents, certain solutes, detergents • Often leads to protein precipitation • The midpoint of the temperature range over which denaturation occurs is called the melting temperature, or Tm. • The abruptness of the change suggests that unfolding is a cooperative process: loss of structure in one part of the protein destabilizes other parts. Under most conditions, denatured proteins exist in a set of partially folded states. Amino Acid Sequence Determines Tertiary Structure • Renaturation = process by which certain denatured globular proteins regain their native structure and biological activity • Anfinsen experiment showed the amino acid sequence contains all the information required to fold the chain. • Even though all proteins have the potential to fold into their native structure, many require some assistance Renaturation of unfolded, denatured ribonuclease. Polypeptides Fold Rapidly by a Stepwise Process • Local secondary structures fold first • Ionic interactions play an important role • Longer range interactions follow • Hydrophobic effect plays a significant role • Process continues until the entire polypeptide folds It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest energy one is found (Levinthal’s Paradox) Some Proteins Undergo Assisted Folding • Chaperone proteins = facilitate correct folding pathways or ideal microenvironments • Hsp70 = bind to hydrophobic regions • Chaperonins = required for the folding of proteins that do not fold spontaneously (Hsp60 in eukaryoyes) Some Folding Pathways Require Isomerization Reactions • Protein disulfide isomerase (PDI) = catalyzes interchange, or shuffling, of disulfide bonds • Peptide prolyl cis-trans isomerase (PPI) = catalyzes the interconversion of the cis and trans isomers of peptide bonds formed by pro residues Defects in Protein Folding Are the Molecular Basis for Many Human Genetic Disorders • Amyloid fiber = protein secreted in a misfolded state and converted to an insoluble extracellular fiber • Amyloidose diseases: type 2 diabetes, alzheimer disease, huntington disease, and parkinson disease • Formation of Disease-Causing Amyloid Fibrils • Native = high degree of β-sheet structure • Misfolded β amyloid promotes aggregation, forming an amyloid fibril Neurodegenerative Conditions • Alzheimer disease = associated with extracellular amyloid deposition by neurons, involving the amyloid-β peptide • Parkinson disease = misfolded form α-synuclein aggregates into spherical filamentous masses called Lewy bodies • Huntington disease = involves the intracellular aggregation of huntingtin, a protein with long polyglutamine repeat Cystic Fibrosis • Cystic fibrosis = caused by defects in the membrane-bound protein cystic fibrosis transmembrane conductance regulator (CFTR) • Deletion of a phe residue causes improper protein folding Death by Misfolding: The Prion Diseases • Prion protein (PRP) = misfolded brain protein Pyramidal cells in the human cerebral cortex Comparable section from a patient with Creutzfeldt-Jakob disease Determination of Protein and Biomolecular Structures Determining Protein Structures • Structural biology = study of three-dimensional structures of biomolecules, including proteins, nucleic acids, lipid membranes, and oligosaccharides • Employs biochemical approaches, physical tools, and computational methods