Proteins: Structure and Function

Proteins Cellular Overview Core Topics Functions Key Properties Amino Acids: properties, classifications, pI Primary Structure, Secondary Structure, and Motifs Tertiary Structure Fibrous vs. Globular Quaternary Structure

Amazing Proteins: Function Catalysts (Enzymes) Transport & Storage The largest class of proteins, accelerate rates of reactions DNA Polymerase Catalase CK2 Kinase Ovalbumin Casein Ion channels Hemoglobin Serum albumin

Amazing Proteins: Function Structural Generate Movement Collagen Keratin Silk Fibroin Actin Myosin

Amazing Proteins: Function Regulation of Metabolism and Gene Expression Protection Insulin Lac repressor Thrombin and Fibrinogen Ricin Immunoglobulin Venom Proteins

Amazing Proteins: Function Signaling and response (inter and intracellular) Apoptosis Membrane proteins Signal transduction

Amazing Proteins: Properties Biopolymers of amino acids Contains a wide range of functional groups Can interact with other proteins or other biological macromolecules to form complex assemblies Some are rigid while others display limited flexibility

a-Amino Acids: Protein Building Blocks R-group or side-chain a-amino group Carboxyl group a-carbon

Amino acids are zwitterionic “Zwitter” = “hybrid” in German Fully protonated forms will have specific pKa’s for the different ionizable protons Amino acids are amphoteric (both acid and base)

Stereochemistry of amino acids

Stereochemistry of amino acids (AA) AA’s synthesized in the lab are racemic mixtures. AA’s from nature are “L” isomers These are all optically-active except for glycine (why?)

Synthesis of Proteins + H2O

Synthesis of Proteins

Synthesis of Proteins N-Terminal End C-Terminal End

Synthesis of Proteins ≠ =

Synthesis of Proteins ≠ =

Synthesis of Proteins ≠ ≠

COMMON AMINO ACIDS 20 common amino acids make up the multitude of proteins we know of

Amino Acids With Aliphatic Side Chains

Amino Acids With Aliphatic Side Chains

Amino Acids With Aliphatic Side Chains

Amino Acids With Aromatic Side Chains

Amino Acids with Aromatic Side Chains Can Be Analyzed by UV Spectroscopy

Amino Acids With Hydroxyl Side Chains

Amino Acid with a Sulfhydryl Side Chain

Disulfide Bond Formation

Amino Acids With Basic Side Chains

Amino Acids With Acidic Side Chains and Their Amide Derivatives

There are some important uncommon amino acids we shall still encounter later on.

pH and Amino Acids Net charge: +1 Net charge: 0 Net charge: -1

Characteristics of Acidic and Basic Amino Acids Acidic amino acids Low pKa Negatively charged at physiological pH Side chains with –COOH Predominantly in unprotonated form Basic amino acids High pKa Function as bases at physiological pH Side chains with N

Protein Structure We use different “levels” to fully describe the structure of a protein.

Primary Structure Amino acid sequence Standard: Left to Right means N to C-terminal Eg. Insulin (AAA40590) The info needed for further folding is contained in the 1o structure. MAPWMHLLTVLALLALWGPNSVQAYSSQHLCGSNLVEALYMTCGRSGFYRPHDRRELEDLQVEQAELGLEAGGLQPSALEMILQKRGIVDQCCNNICTFNQLQNYCNVP

Secondary Structure The regular local structure based on the hydrogen bonding pattern of the polypeptide backbone α helices β strands (β sheets) Turns and Loops WHY will there be localized folding and twisting? Are all conformations possible?

α Helix First proposed by Linus Pauling and Robert Corey in 1951. 3.6 residues per turn, 1.5 Angstroms rise per residue Residues face outward

α Helix α-helix is stabilized by H-bonding between CO and NH groups Except for amino acid residues at the end of the α-helix, all main chain CO and NH are H-bonded

α Helix representation

β strand Fully extended β sheets are formed by linking 2 or more strands by H-bonding Beta-sheet also proposed by Corey and Pauling in 1951.

PARALLEL ANTIPARALLEL

The Beta Turn (aka beta bend, tight turn) allows the peptide chain to reverse direction carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away proline and glycine are prevalent in beta turns

Mixed β Sheets

Twisted β Sheets

 Loops

What Determines the Secondary Structure? The amino acid sequence determines the secondary structure The α helix can be regarded as the default conformation – Amino acids that favor α helices: Glu, Gln, Met, Ala, Leu – Amino acids that disrupt α helices: Val, Thr, Ile, Ser, Asx, Pro

What Determines the Secondary Structure? Branching at the β-carbon, such as in valine, destabilizes the α helix because of steric interactions Ser, Asp, and Asn tend to disrupt α helices because their side chains compete for H-bonding with the main chain amide NH and carbonyl Proline tends to disrupt both α helices and β sheets Glycine readily fits in all structures thus it does not favor α helices in particular

Can the Secondary Structure Be Predicted? Predictions of secondary structure of proteins adopted by a sequence of six or fewer residues have proved to be 60 to 70% accurate Many protein chemists have tried to predict structure based on sequence Chou-Fasman: each amino acid is assigned a "propensity" for forming helices or sheets Chou-Fasman is only modestly successful and doesn't predict how sheets and helices arrange George Rose may be much closer to solving the problem. See Proteins 22, 81-99 (1995)

Modeling protein folding with Linus (George Rose)

Tertiary Structure The overall 3-D fold of the polypeptide chain The amino acid sequence determines the tertiary structure (Christian Anfinsen) The polypeptide chain folds so that its hydrophobic side chains are buried and its polar charged chains are on the surface Exception : membrane proteins Reverse : hydrophobic out, hydrophilic in A single polypeptide chain may have several folding domains Stabilized by H-bonding, LDF, noncovalent interactions, dipole interactions, ionic interactions, disulfide bonds

Fibrous and Globular Proteins

Fibrous Proteins Much or most of the polypeptide chain is organized approximately parallel to a single axis Fibrous proteins are often mechanically strong Fibrous proteins are usually insoluble Usually play a structural role in nature

Examples of Fibrous Proteins Alpha Keratin: hair, nails, claws, horns, beaks Beta Keratin: silk fibers (alternating Gly-Ala-Ser)

Examples of Fibrous Proteins Collagen: connective tissue- tendons, cartilage, bones, teeth Nearly one residue out of three is Gly Proline content is unusually high Unusual amino acids found: (4-hydroxyproline, 3- hydroxyproline , 5- hydroxylysine) Special uncommon triple helix!

Globular Proteins Most polar residues face the outside of the protein and interact with solvent Most hydrophobic residues face the interior of the protein and interact with each other Packing of residues is close but empty spaces exist in the form of small cavities Helices and sheets often pack in layers Hydrophobic residues are sandwiched between the layers Outside layers are covered with mostly polar residues that interact favorably with solvent

An amphiphilic helix in flavodoxin: A nonpolar helix in citrate synthase: A polar helix in calmodulin:

Quaternary Structures Spatial arrangement of subunits and the nature of their interactions. Can be hetero and/or homosubunits Simplest example: dimer (e.g. insulin) ADVANTAGES of 4o Structures Stability: reduction of surface to volume ratio Genetic economy and efficiency Bringing catalytic sites together Cooperativity

Protein Folding CHAPERONES assist protein folding The largest favorable contribution to folding is the entropy term for the interaction of nonpolar residues with the solvent CHAPERONES assist protein folding to protect nascent proteins from the concentrated protein matrix in the cell and perhaps to accelerate slow steps