Protein

Understanding of structural protein at different level (primary, secondary, tertiaryand quaternary);

Mitesh Shrestha

Protein

• Proteins are polymers of amino acids covalently linked through peptide bonds into a chain.

• There are 20 different amino acids that make up essentially all proteins on earth.

• Each of these amino acids has a fundamental design composed of a central carbon (also called the alpha carbon) bonded to: – a hydrogen

– a carboxyl group

– an amino group

– a unique side chain or R-group

Protein

• Thus, the characteristic that distinguishes one amino acid from another is its unique side chain, and it is the side chain that dictates an amino acids chemical properties.

• The unique side chains confer unique chemical properties on amino acids, and dictate how each amino acid interacts with the others in a protein.

• Amino acids can thus be classified as being hydrophobic versus hydrophilic, and uncharged versus positively-charged versus negatively-charged.

Understanding of structural protein at different level

• Primary

• Secondary

• Tertiary

• Quaternary

Biology/Chemistry of Protein Structure

Primary

Secondary

Tertiary

Quaternary

Assembly

Folding

Packing

Interaction

S T

R U

C T

U R

E

P R

O C

E S S

Protein Assembly

• occurs at the ribosome

• involves dehydration synthesis and polymerization of amino acids attached to tRNA:

NH - {A + B A-B + H O} -COO

• thermodynamically unfavorable, with E = +10kJ/mol, thus coupled to reactions that act as sources of free energy

• yields primary structure

2 n 3

+ -

Primary structure

• The primary structure of a protein refers to the linear sequence of amino acids in the polypeptide chain.

• The primary structure is held together by covalent bonds such as peptide bonds, which are made during the process of protein biosynthesis or translation.

• The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity.

Primary structure

• It is strictly recommended to use the words "amino acid residues" when discussing proteins because when a peptide bond is formed, a water molecule is lost, and therefore proteins are made up of amino acid residues.

• Post-translational modification such as phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read from the gene.

Primary structure

• The minimum size of a protein is defined as about 50 residues; smaller chains are referred to simply as peptides.

• So the primary structure of a small protein would consist of a sequence of 50 or so residues.

• There is no theoretical maximum size, but the largest protein so far discovered has about 30,000 residues.

• Since the average molecular weight of a residue is about 110 Da, that single chain has a molecular weight of over 3 million Daltons.

Primary structure

Primary Structure

• linear

• ordered

• 1 dimensional

• sequence of amino acid polymer

• by convention, written from amino end to carboxyl end

• a perfectly linear amino acid polymer is neither functional nor energetically favorable folding!

primary structure of human insulin

CHAIN 1: GIVEQ CCTSI CSLYQ LENYC N

CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT

Protein Folding

• tumbles towards conformations that reduce E (this process is thermo-dynamically favorable)

• yields secondary structure

• occurs in the cytosol

• involves localized spatial interaction among primary structure elements, i.e. the amino acids

• may or may not involve chaperone proteins

Secondary Structure

• Protein secondary structure is the general three-dimensional form of local segments of proteins.

• Secondary structure can be formally defined by the pattern of hydrogen bonds of the protein (such as alpha helices and beta sheets) that are observed in an atomic-resolution structure.

• More specifically, the secondary structure is defined by the patterns of hydrogen bonds formed between amine hydrogen and carbonyl oxygen atoms contained in the backbone peptide bonds of the protein.

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Secondary Structure • The chemical nature of the carboxyl and amino groups of all amino

acids permit hydrogen bond formation (stability) and hence defines secondary structures within the protein.

• The R group has an impact on the likelihood of secondary structure formation (proline is an extreme case)

• This leads to a propensity for amino acids to exist in a particular secondary structure conformation

• Helices and sheets are the regular secondary structures, but irregular secondary structures exist and can be critical for biological function

Alpha Helix

• A helix can turn right or left from N to C terminus – only right-handed are observed in nature as this produces less clashes

• All hydrogen bonds are satisfied except at the ends = stable

Alpha Helix

Alpha Structure Features

• 3.6 residues per turn

• 5.4 angstroms in length per turn

• carboxyl group of residue i hydrogen bonds to amino group of residue i+4

Other (Rarer) Helix Types - 310

• Less favorable geometry

• 3 residues per turn with i+3 not i+4

• Hence narrower and more elongated

• Usually seen at the end of an alpha helix

Other (Very Rare) Helix Types - π

• Less favorable geometry

• 4 residues per turn with i+5 not i+4

• Squat and constrained

Helix Structures

Φ ψ H Bond R/t A/t Alpha -57.8 -47 i, i + 4 3.6 13 3-10 Helix -49 -26 i, i + 3 3.0 10 Pi Helix -57 -80 i , i + 5 4.4 16

Beta Sheets

Beta Sheets Continued

• Between adjacent polypeptide chains

• Phi and psi are rotated approximately 180 degrees from each other

• Mixed sheets are less common

• Viewed end on the sheet has a right handed twist that may fold back upon itself leading to a barrel shape (a beta barrel)

• Beta bulge is a variant; residue on one strand forms two hydrogen bonds with residue on other – causes one strand to bulge – occurs most frequently in parallel sheets

Other Secondary Structures – Loop or Coil

• Often functionally significant

• Different types – Hairpin loops (aka reverse turns) – often between

anti-parallel beta strands

– Omega loops – beginning and end close (6-16 residues)

– Extended loops – more than 16 residues

Beta Sheet Features

• Sheets can be made up of any number of strands

• Orientation and hydrogen bonding pattern of strands gives rise to flat or twisted sheets

• Parallel sheets buried inside, while Antiparallel sheets occurs on the surface

Beta Sheet

More Beta Structures

Beta Bulge chymotrypsin (1CHG.PDB) involving residues 33 and 41-42 Anti parallel

Beta Twist pancreatic trypsin inhibitor (5PTI) 0 to 30 degrees per residue Distortion of tetrahedral N atom

Definition of -turn

A -turn is defined by four consecutive residues i, i+1, i+2 and i+3

that do not form a helix and have a C(i)-C(i+3) distance less than 7Å and the turn lead to reversal in the protein chain. (Richardson, 1981).

The conformation of -turn is defined in terms of and of two central residues, i+1 and i+2 and can be classified into different types on the basis of and .

i

i+1 i+2

i+3 H-bond

D <7Å

Beta turns

i + 1 Pro

i + 2 Pro or Gly

i + 3 Gly

Interactions

• Covalent bonds

Disulphide bond (2.2 0A) between two Cys residues

• Non-covalent bonds

Long range electrostatic interaction

Short range (4 0A) van der Waals interaction

Hydrogen bond (3 0A)

Secondary Structure • non-linear

• 3 dimensional

• localized to regions of an amino acid chain

• formed and stabilized by hydrogen bonding, electrostatic and van der Waals interactions

Ramachandran Plot

• Pauling built models based on the following principles, codified by Ramachandran:

(1) bond lengths and angles – should be similar to those found in individual amino acids and small peptides

(2) peptide bond – should be planer

(3) overlaps – not permitted, pairs of atoms no closer than sum of their covalent radii

(4) stabilization – have sterics that permit hydrogen bonding

• Two degrees of freedom:

(1) (phi) angle = rotation about N – C

(2) (psi) angle = rotation about C – C

• A linear amino acid polymer with some folds is

better but still not functional nor completely energetically favorable packing!

Protein Packing

• occurs in the cytosol (~60% bulk water, ~40% water of hydration)

• involves interaction between secondary structure elements and solvent

• may be promoted by chaperones, membrane proteins

• tumbles into molten globule states

• overall entropy loss is small enough so enthalpy determines sign of E, which decreases (loss in entropy from packing counteracted by gain from desolvation and reorganization of water, i.e. hydrophobic effect)

• yields tertiary structure

Tertiary Structure

• The term protein tertiary structure refers to a protein's geometric shape.

• The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains.

• Amino acid side chains may interact and bond in a number of ways. The interactions and bonds of side chains within a particular protein determine its tertiary structure.

• The protein tertiary structure is defined by its atomic coordinates. These coordinates may refer either to a protein domain or to the entire tertiary structure.

Aspects which determine tertiary structure

• Covalent disulfide bonds from between closely aligned cysteine residues form the unique Amino Acid cystine.

• Nearly all of the polar, hydrophilic R groups are located in the surface, where they may interact with water

• The nonpolar, hydropobic R groups are usually located inside the molecule

Motifs and Domains

• Motif – a small structural domain that can be recognized in a variety of proteins

• Domain – Portion of a protein that has a tertiary structure of its own. In larger proteins each domain is connected to other domains by short flexible regions of polypeptide.

Tertiary Structure as Dictated by the Environment

• Proteins exist in an aqueous environment where hydrophilic residues tend to group at the surface and hydrophobic residues form the core – but the backbone of all residues is somewhat hydrophilic – therefore it is important to have this neutralized by satisfying all hydrogen bonds as is achieved in the formation of secondary structures

• Polar residues must be satisfied in the same way – on occasion pockets of water (discreet from the solvent) exist as an intrinsic part of the protein to satisfy this need

• Ion pairs (aka salt bridge) form important interactions

• Disulphide linkages between cysteines form the strongest (ie covalent tertiary linkages); the majority of cysteines do not form such linkages

Tertiary Structure as Dictated by Protein Modification

• To the amino acid itself eg hydroxyproline needed for collagen formation

• Addition of carbohydrates (intracellular localization)

• Addition of lipids (binding to the membrane)

• Association with small molecules – notably metals eg hemoglobin

Tertiary Structure

• non-linear

• 3 dimensional

• global but restricted to the amino acid polymer

• formed and stabilized by hydrogen bonding, covalent (e.g. disulfide) bonding, hydrophobic packing toward core and hydrophilic exposure to solvent

• A globular amino acid polymer folded and compacted is somewhat functional (catalytic) and energetically favorable interaction!

Protein Interaction

• occurs in the cytosol, in close proximity to other folded and packed proteins

• involves interaction among tertiary structure elements of separate polymer chains

• may be promoted by chaperones, membrane proteins, cytosolic and extracellular elements as well as the proteins’ own propensities

• E decreases further due to further desolvation and reduction of surface area • globular proteins, e.g. hemoglobin, largely involved in catalytic roles

• fibrous proteins, e.g. collagen,

largely involved in structural roles

• yields quaternary structure

PHAR201 Lecture 1 2012 48

Quaternary Structure

• The biological function of some molecules is determined by multiple polypeptide chains – multimeric proteins

• Chains can be identical eg homeodimer or different eg heterodimer

• The interactions within multimers is the same as that found in tertiary and secondary structures

Quaternary Structure

• Not all proteins have a quaternary structure

• A composite of multiple poly-peptide chains is called an oligomer or multimeric

• Hemoglobin is an example of a tetramer

Quaternary Structure

• non-linear

• 3 dimensional

• global, and across distinct amino acid polymers

• formed by hydrogen bonding, covalent bonding, hydrophobic packing and hydrophilic exposure

• favorable, functional structures occur frequently and have been categorized

Levels of Description of Structural Complexity

• Primary Structure (AA sequence)

• Secondary Structure – Spatial arrangement of a polypeptide’s backbone atoms

without regard to side-chain conformations • , , coil, turns (Venkatachalam, 1968)

– Super-Secondary Structure • , , /, + (Rao and Rassman, 1973)

• Tertiary Structure – 3-D structure of an entire polypeptide

• Quarternary Structure – Spatial arrangement of subunits (2 or more polypeptide

chains)

Protein structure: overview

Structural element Description

primary structure amino acid sequence of protein

secondary structure helices, sheets, turns/loops

super-secondary structure association of secondary structures

domain self-contained structural unit

tertiary structure folded structure of whole protein • includes disulfide bonds

quaternary structure assembled complex (oligomer) • homo-oligomeric (1 protein type) • hetero-oligomeric (>1 type)

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Upcoming Lecture

• Conformations of the poly peptide chain, helix-coil transitions, the protein globule and hydrophobic interactions;

• The structure and stability of the globule, antibody and antigens, fibrous proteins.

Assignment

• Write about structural organization of proteins ? [8]

• Draw the structure of all the amino acids. [7]

• Write about Ramachandran plot. [5]