Biochemistry Sixth Edition Chapter 2 Protein Composition and Structure Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer

Protein composition and structure Linear polymer of amino acids – folding – 3D structure Chapter 2. Proteins contain various functional groups Proteins interact with one another and with other macromolecules to form complex assemblies Some rigid, some flexible The most versatile macromolecules

Structure dictates function.

Insect flight tissue

Flexibility & function: lactoferrin

2.1 Proteins are built from a repertoire of 20 amino acids  -carbon, side chain, chiral only L isomers, S absolute configuration Zwitterions (dipolar ions) at neutral pH 20 amino acids with different size, charge, shape, hydrogen bonding capacity, hydrophobic character, chemical reactivity

Isomers of amino acids

S absolute configuration

Ionization of amino acids

achiral

Aliphatic side chains thioether2 chiral centers

Proline has a ring structure.

Aromatic side chains

hydroxyl group indole ring

chiral carbon

carboxamide

Thiol or sulfhydryl

Positively charged, basic

Histidine ionization

acidic

Asn, Gln, Ile, Trp R, N, D, Q, E, K, F, W, Y

Reason for selection of 20 aa 1.Diverse 2.Available from prebiotic reactions 3.Not very reactive

2.2 Primary structure: amino acids are linked by peptide bonds to form polypeptide chains Peptide bond Oligopeptide, polypeptide, protein, residue, polarity YGGFL vs. LFGGY Main chain or backbone vs. Side chain disulfide bonds

Amide bond Kinetically stable

rich in hydrogen-bonding potential 50-2,000 aa Average MW; 110

Disulfide bond Less disulfide bonds in intracellular proteins, Reason to add DTT,  -ME, etc

Proteins have unique amino acid sequences that are specified by genes Each protein has a unique, precisely defined amino acid sequence Sequence information: mechanism of action, three dimensional structure, molecular pathology, evolutionary history

Bovine insulin

Polypeptide chains are flexible yet conformationally restricted peptide bond is planar and has double bond character

Peptide bonds are planar.

Typical bond lengths

Almost all peptide bonds are trans

X-Pro: cis and trans

Angle of rotation about the bond between N and C  : phi,  Angle of rotation about the bond between C  and carbonyl carbon: psi,  Limited freedom of rotation by steric exclusion

Ramachandran diagram Rigidity and restricted set of allowed angles limit the number of structures accessible to unfolded form sufficiently to allow proteins to fold

2.3 Secondary structure: polypeptide chains can fold into regular structures such as the alpha helix, the beta sheet, and turns and loops Folding into a regularly repeating structure Linus Pauling and Robert Corey in 1951

The alpha helix is a coiled structure stabilized by intrachain hydrogen bonds only main chain Almost all the main chain CO and NH groups are H-bonded. rodlike shape

alpha helix rise of 1.5A, 3.6 aa per turn, pitch=1.5A*3.6=5.4A

hydrogen bonds between NH and CO

Right-handed helices are energetically more favorable

Ferritin Less than 10 helical turns (~45A)

Beta sheets are stabilized by hydrogen bonding between polypeptide strands Beta strand is almost fully extended Beta sheet: H bond between strands rise of 3.5A Parallel or antiparallel

Almost fully extended side chain

antiparallel beta sheet

parallel beta sheet

mixed

Fatty acid binding protein

Polypeptide chains can change direction by making reverse turns and loops Beta turn and omega loop Loops do not have periodic structure but usually well defined on the surface of proteins

Beta turn: CO of residue i H-bonded to NH of residue i+3

Loops on a protein surface

Fibrous proteins provide structural support for cells and tissues  -Keratin (hair):  coiled coil, left-handed superhelix Heptad repeats  3.5 residues/turn  7 residues/2 turns Stability: hydrophobic and ionic interactions, disulfide bonds Collagen: three helical chains, Gly-Pro-Hyp Stability: hydrogen bonds, steric repulsion of pyrrolidine rings

2.4 Tertiary structure: water-soluble proteins fold into compact structure with nonpolar cores Main chain NH and CO groups are maximally H-bonded in the core Thermodynamically most stable Side chain and main chain Tertiary structure

Myoglobin: compact

Distribution of side chains surfacecross-section The interior consists almost entirely of nonpolar residues The outside consists of both polar and nonpolar residues

membrane proteins like porins

Motif or supersecondary structure

Domain: compact globular unit CD4

2.5 Quaternary structure: polypeptide chains can assemble into mutisubunit structures Quaternary structure: the spatial arrangement of subunits and the nature of their interactions

Cro protein of bacteriophage lambda

hemoglobin

rhinovirus particle

2.6 The amino acid sequence of a protein determines its three dimensional structure Refolding of ribonuclease Christian Anfisen After denaturation, what conditions were required to restore the structure Sequence specifies conformatiom and function

Chaotropic agent (urea, gunidinium chloride): non-covalent bond breaker Reducing agent

Bovine ribonuclease

Reducing agent (beta-mercaptoethanol): S-S to SH

Oxidation in 8M urea And dialysis Thermodynamically preferred structure

Amino acids have different propensities for forming alpha helices, beta sheets, and beta turns Secondary structure: only main chain Effects of side chains on secondary structure Branching at  -carbon as in Val, Thr, Ile:  sheet H-bonding capability of Ser, Asp, Asn: turn Pro, Gly: turn

Tertiary interactions may be decisive in determining the secondary structure Alternative conformation of VDLLKN

Protein misfolding and aggregation are associated with some neurological diseases Bovine spongiform encephalopathy (mad cow disease) Creutzfeldt-Jakob disease (CJD) scrapie Prions 1.The transmissible agent consists of aggregated forms of a protein 2.Resistant to treatments with agents that degrade most proteins 3.Derived from PrP that is normally present in the brain Some parts of PrP in  -helix or turn are converted to  -strand   sheets Protein-only model for prion disease transmission Alzheimer disease – amyloid precursor protein(APP)  A    amyloid plaque

Protein folding is a highly cooperative process All or none process from a cooperative transition

Transition from foled to unfolded state

Partially denatured protein solution

Proteins fold by progressive stabilization of intermediates rather than by random search Levinthal’s paradox: enormous difference between calculated and actual folding times Typing monkey Retention of partly correct intermediates

Typing monkey analogy

Prediction of three-dimensional structure from sequence remains a great challenge ab initio prediction knowledge-based methods

Protein modification and cleavage confer new capability Acetylation of N-termini, Hydroxylation of proline, Carboxylation of glutamate, Glycosylation, Phosphoryation of Ser, Thr, Tyr, Green fluorescent protein (GFP) Cleavage

Space-filling model

Ball-and-stick model

Backbone model

Ribbon diagram