Biochemistry 301 Principles of Protein Structure Walter Chazin 5140 BIOSCI/MRBIII Walter.Chazin Jan. 8-10, 2003

Text Books Branden and Tooze Introduction to Protein Structure Voet, Voet and Pratt Fundamentals of Biochemistry Stryer Biochemistry

Proteins: Polymers of Amino Acids 20 different amino acids: many combinations Proteins are made in the RIBOSOME

Amino Acid Chemistry NH 2 CC R1R1 CO H NHCC R2R2 COOH H NH 2 CC R COOH H amino acid 20 different types Amino acid PolypeptideProtein NH 2 CC R1R1 COOH H NH 2 CC R2R2 COOH H

Amino Acid Chemistry NH 2 CC R COOH H amino acid The free amino and carboxylic acid groups have pKa’s COOH COO - pKa ~ 2.2 NH 2 NH 3 + pKa ~ 9.4 At physiological pH, amino acids are zwitterions + NH 3 CC R COO - H

Amino Acid Chemistry Note the axes Also titratable groups in side chain

GlycineGly - G AlanineAla - A ValineVal - V LeucineLeu - L IsoleucineIle - I Amino Acids with Aliphatic R-Groups pKa’s

Amino Acids with Polar R-Groups Non-Aromatic Amino Acids with Hydroxyl R-Groups SerineSer - S ~13 ThreonineThr - T ~13 Amino Acids with Sulfur-Containing R-Groups CysteineCys - C MethionineMet-M

Aspartic AcidAsp - D AsparagineAsn - N Glutamic AcidGlu - E GlutamineGln - Q Acidic Amino Acids and Amide Conjugates

Basic Amino Acids ArginineArg - R LysineLys - K HistidineHis - H

Aromatic Amino Acids and Proline PhenylalaninePhe - F TyrosineTyr - Y TryptophanTrp-W ProlinePro - P

Hierarchy of Protein Structure 20 different amino acids: many combinations The order of amino acids: Protein sequence Primary Structure Local conformation, depends on sequence Secondary Structure Overall structure of the chain(s) in full 3D Tertiary/Quaternary Structure

Beyond Primary Structure: The Peptide Bond -C - N- O = - H -C = N- O-O- - - H Resonance structures  Peptide plane is flat  angle ~180º  Partial double-bond: Peptide bond

Implications of Peptide Planes   angle varies little,  and  angles vary alot  Many  /  combinations cause atoms to collide  Each residue is sandwiched between two planes CC HR   Peptide planes CC H R CC

Polypeptide Backbone  Backbone restricted  limited conformations  Collisions with side chain groups further limit  /  combinations CC HR   CC HR CC HR

Secondary Structure Local Conformation of Consecutive Residues Three low energy backbone  combinations 1. Right-hand helix:  -helix (-40°, -60°) 2. Extended: antiparallel  -sheet (140°, -140°) rare 3. Left-hand helix (rare):  -helix (45°, 45°)  Glycine: special it has no side chain! Hydrogen bonds between backbone atoms provides stability to secondary structures Amino acids have specific preferences

Secondary Structure-  Helix H-bond

Secondary Structure-  Sheet OxygenNitrogen R Group Hydrogen Carbon  Carbonyl C H Bond

Secondary Structure-  Turn  Reverses direction of the chain

Ribbon and Topology Diagrams Representations of Secondary Structures  Sheets (arrows), Helices (cylinders) B/T- Figure 2.17

Ribbon and Topology Diagrams Organization of Secondary Structures helix B/T- Figure 2.11

Beyond Secondary Structure Supersecondary structure (motifs): small, discrete, commonly observed aggregates of secondary structures   sheet   helix-loop-helix   Domains: independent units of structure   barrel  four-helix bundle *Domains and motifs sometimes interchanged*

Protein Motifs V/V/P- Figure 6.28

Hairpin Motif B/T- Figure 2.14

Helix-Loop-Helix (H-L-H) Motif B/T- Figure 2.12

EF-Hand H-L-H Motif B/T- Figure 2.13

Greek Key Motif B/T- Figure 2.15

Multi-Domain (Modular) Proteins EGF Protease Kringle Ca-binding Protein Domain

Tertiary Structure Definition: Overall 3D form of a molecule  Organization of the secondary structures/ motifs/domains  Optimization of interactions between residues  A specific 3D structure is formed All proteins have multiple secondary structures, almost always multiple motifs, and in some cases multiple domains

Tertiary Structure Specific structures result from long-range interactions  Electrostatic (charged) interactions  Hydrogen bonds (O  H, N  H, S  H)   Hydrophobic interactions Soluble proteins have an inside (core) and outside  Folding driven by water- hydrophilic/phobic  Side chain properties specify core/exterior   Some interactions inside, others outside

Tertiary Structure I. Ionic Interactions (exterior) Forms between 2 charged side chains: 1 Negative – Glu,Asp 1 Positive – Lys,Arg,His  Also called “salt bridges”.  Ionic interactions are pH-dependent (pKa).  Occurs at the exterior  NOTE: pKs for in the interior of a protein may be very different from free amino acid.

Tertiary Structure II. Hydrogen bonds (interior and exterior) Forms between side chains/backbone/water: Charged side chains: Glu,Asp,His,Lys,Arg Polar chains: Ser,Thr,Cys,Asn,Gln,[Tyr,Trp]  Not a specific covalent bond – lower energy.  Occurs inside, at the exterior, and with water.

Tertiary Structure III. Hydrophobic Interactions (interior) Forms between side chains of non-polar residues: Aliphatic (Ala,Val,Leu,Ile,Pro,Met) Aromatic (Phe,Trp,[Tyr])  Clusters of side chains- but no requirement for a specific orientation like an H-bond  In the protein interior, away from water  Not pH dependent

Tertiary Structure IV. Disulfide Bonds (interior and exterior) Forms between Cys residues: Cys-SH + HS-Cys  Cys-S-S-Cys  Catalyzed by specific enzymes, oxidizing agents  Restricts flexibility of the protein  Usually within a protein, less for linking proteins

Disulfide Bonding V/V/P- Figure 16.6

Quaternary Structure Definition: Organization of multiple chain associations  Oligomerization- Homo (self), Hetero (different)   Used in organizing single proteins and protein machines Specific structures result from long-range interactions  Electrostatic (charged) interactions  Hydrogen bonds (O  H, N  H, S  H)   Hydrophobic interactions  Disulfides only VERY infrequently

Quaternary Structure The classic example- hemoglobin  2 -  2 B/T- Figure 3.7 END OF PART 1

Protein Structure from Sequence The pattern of amino acid side chains determines the local conformation and the global structure *Pattern is more important than exact sequence* A T V R L L E W E D L Reporting/Comparing Protein Sequences A T V R L L E Y K D L 5 10 h-CaM b-CaM conservativenon-conservative

Proteins Fold To Their Native Structure Folded proteins are only marginally stable!!  ~0.4 kJmol -1 required to unfold (cf. ~20/H-bond)  Balance loss of entropy vs. stabilizing forces Protein fold is specified by sequence  Reversible reaction- denature (fold)/renature   Even single mutations can cause changes  Recent discovery that amyloid diseases (eg. CJD, Alzheimer) are due to unstable protein folding

How Does a Protein Find It’s Fold? A protein of n residues: 20 n possible sequences! 100 residue protein has possibilities 1.3 X !  The latest estimates indicate < 40,000 sequences in the human genome  THERE MUST BE RULES! 20 different amino acids: many combinations NC 1234 Amino terminusCarboxyl terminus Residue number

Limitations on Protein Sequence  Minimum length based on ability to perform a biochemical function: ~40 residues (e.g. inhibitors)  Maximum length based on complexity of assembly: Conversion of DNA code and production of proteins is carried out by molecular machines that are not perfect. If the sequence gets too long, too many errors will build up. *Length is generally residues*

Protein Folding The hydrophobic effect is the major driving force  Hydrophobic side chains cluster/exclude water  Release of water cages in unfolded state Other forces providing stability to the folded state  Hydrogen bonds   Electrostatic interactions  Chemical cross links- Disulfides, metal ions

Protein Folding Random folding has too many possibilities Backbone restricted but side chains not A 100 residue protein would require s to search all conformations (age of universe < s) Most proteins fold in less than 10 s!!  Proteins must fold along specific pathways!!

Protein Folding Pathways Usual order of folding events  Secondary structures formed quickly (local)  Secondary structures aggregate to form motifs  Hydrophobic collapse to form domains  Coalescence of domains Molecular chaperones assist folding in-vivo  Complexity of large chains/multi-domains  Cellular environment is rich in interacting molecules  Chaperones sequester proteins and allow time to fold

Progressive Folding of Proteins From Disordered to Native State Protein Folding Funnel V/V/P- Figures 6.37/38

Functional Classes of Proteins Receptors- sense stimuli, e.g. in neurons Channels- control cell contents Transport- e.g. hemoglobin in blood Storage- e.g. ferritin in liver Enzyme- catalyze biochemical reactions Cell function- multi-protein machines Structural- collagen in skin Immune response- antibodies

Structural Classes of Proteins 1. Globular proteins (enzymes, molecular machines)  Variety of secondary structures  Approximately spherical shape  Water soluble  Function in dynamic roles (e.g. catalysis, regulation, transport, gene processing)

Globular Proteins V/V/P- Figure 6.27 Hemoglobin  Conconavalin A Triose Phosphate isomerase

Structural Classes of Proteins 2. Fibrous Proteins (fibrils, structural proteins)  One dominating secondary structure  Typically narrow, rod-like shape  Poor water solubility  Function in structural roles (e.g. cytoskeleton, bone, skin)

Collagen: A Fibrous Protein V/V/P- Figures 6.17/18 Triple Helix Gly-Pro-Pro Repeat Stabilizing Inter-strand H-bonds

Structural Classes of Proteins 3. Membrane Proteins (receptors, channels)  Inserted into (through) membranes  Multi-domain- membrane spanning, cytoplasmic, and extra-cellular domains  Poor water solubility  Function in cell communication (e.g. cell signaling, transport)

Photosynthetic Reaction Center B/T Figure 13.6 Extracellular Intracellular (cytoplasmic) Membrane- spanning

In the physical sense, the progression of living organisms results from the communication between molecules. Interaction between molecules is determined by binding affinities.

Binding Classification of Proteins Structural- other structural proteins Receptors- regulatory proteins, transmitters Toxins- receptors Transport- O 2 /CO 2, cholesterol, metals, sugars Storage- metals, amino acids, Enzymes- substrates, inhibitors, co-factors Cell function- proteins, RNA, DNA, metals, ions Immune response- foreign matter (antigens)

Surface Determines What Binds  Steric access  Shape  Hydrophobic accessible surface  Electrostatic surface Sequence and structure optimized to generate surface properties for requisite binding event(s)

Determinants of Protein Surface Function requires specific amino acid properties  Not all amino acids are equally useful  Abundant: Leu, Ala, Gly, Ser, Val, Glu  Rare: Trp, Cys, Met, His Post-translational modifications  Addition of co-factors- metals, hemes, etc.  Chemical modification- phosphorylation, glycosylation, acetylation, ubiquination, sumoylation

Binding Alters Protein Structure Mechanisms of Achieving Functional Properties  Allosteric Control- binding at one site effects changes in conformation or chemistry at a point distant in space  Stimulation/inhibition by control factors- proteins, ions, metals control progression of a biochemical process (e.g. controlling access to active site) 3. Reversible covalent modification- chemical bonding, e.g. phosphorylation (kinase/phosphatase) 4. Proteolytic activation/inactivation- irreversible, involves cleavage of one or more peptide bonds

Calcium Signal Transduction Allostery & Stimulation by Control Factor Target Ca 2+ Calmodulin

Sequence  Structure  Function Many sequences can give same structure  Side chain pattern more important than sequence When homology is high (>50%), likely to have same structure and function (Structural Genomics)  Cores conserved  Surfaces and loops more variable *3-D shape more conserved than sequence* *There are a limited number of structural frameworks*

I. Homologous: similar sequence (cytochrome c)  Same structure  Same function  Modeling structure from homology Varied Relationships Between Sequence, Structure and Function

V/V/P Figure 6.31 C-Type Cytochromes Same structure/function- Different Sequence Heme  Constant structural elements and basic architecture

Varied Relationships Between Sequence, Structure and Function I. Homologous: very similar sequence (cytochrome c)  Same structure  Same function  Modeling structure from homology II. Similar function- different sequence (dehydrogenases)  One domain same structure  One domain different

B/T Figure 10.8 NAD-Binding Domains Conserved Domains/Functional Elements Lactate DehydrogenaseAlcohol Dehydrogenase

Varied Relationships Between Sequence, Structure and Function I. Homologous: very similar sequence (cytochrome c)  Same structure  Same function  Modeling structure from homology II. Similar function- different sequence (dehydrogenases)  One domain same structure  One domain different III. Similar structure- different function (cf. thioredoxin)  Same 3-D structure  Not same function

B/T Figures 10.8/2.7 NADH-Binding and Redox Same structure- Different Function Alcohol DehydrogenaseLactate Dehydrogenase Thioredoxin