protein folding & biospectroscopy f14pfb dr david robinson lecture 2
TRANSCRIPT
Protein Folding & Biospectroscopy
F14PFB
Dr David Robinson
Lecture 2
Principles of protein structure and function
• Function is derived from structure
• Structure is derived from amino acid sequence
• Different activities and shapes of proteins due to different amino acid sequences
A reminder…• Basic Amino Acid
Structure:– The side chain, R,
varies for each ofthe 20 amino acids
C
RR
C
H
NO
OHH
H
Aminogroup
Carboxylgroup
Side chain
The Peptide Bond
• Dehydration synthesis
• Repeating backbone: N–C –C –N–C –C
– Convention – start at amino terminus and proceed to carboxy terminus
O O
Levels of Protein StructureLevels of Protein Structure
- helix
Myoglobin
Hemoglobin
The folded protein structure is stabilized by a variety of weak chemical interaction, and in some cases covalent (disulfide) bonds between cysteine residues
R– CH2–S–S–CH2–R
Cys Cys
Disulfide bond:
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)
Protein structure:Protein structure: overview overview
Primary & Secondary StructurePrimary & Secondary Structure Primary structurePrimary structure = the linear sequence of
amino acids comprising a protein:AGVGTVPMTAYGNDIQYYGQVT…
Secondary structureSecondary structure• Regular patterns of hydrogen bonding in proteins
result in two patterns that emerge in nearly every protein structure known: the -helix and the-sheet
• The location of direction of these periodic, repeating structures is known as the secondary secondary structurestructure of the protein
The alpha helixThe alpha helix 60°
Properties of the alpha helixProperties of the alpha helix 60° Hydrogen bondsHydrogen bonds
between C=O ofresidue n, andNH of residuen+4
3.6 residues/turn 1.5 Å/residue rise 100°/residue turn
Properties of Properties of -helices-helices 4 – 40+ residues in length Often amphipathic or “dual-natured”
• Half hydrophobic and half hydrophilic• Mostly when surface-exposed
If we examine many -helices,we find trends…• Helix formers: Ala, Glu, Leu,
Met• Helix breakers: Pro, Gly, Tyr,
Ser
The beta strand (& sheet)The beta strand (& sheet) 135° +135°
Properties of beta sheetsProperties of beta sheets Formed of stretches of 5-10 residues in
extended conformation Pleated – each C a bit
above or below the previous Parallel/antiparallelParallel/antiparallel,
contiguous/non-contiguous
Parallel and anti-parallel Parallel and anti-parallel -sheets-sheets Anti-parallel is slightly energetically favoured
Anti-parallelAnti-parallel ParallelParallel
Turns and LoopsTurns and Loops Secondary structure elements are connected by
regions of turns and loops Turns – short regions
of non-, non-conformation
Loops – larger stretches with no secondary structure. Often disordered.• “Random coil”• Sequences vary much more than secondary
structure regions
Levels of Protein Levels of Protein StructureStructure
Secondary structure elements combine to form tertiary structure
Quaternary structure occurs in multienzyme complexes• Many proteins are
active only as homodimers, homotetramers, etc.
Protein Folding• Forming polypeptide chain requires energy
and information (template) – ie translation from RNA protein SEQUENCE
Protein Folding• Forming polypeptide sequence requires
energy and information (template)
• Forming native conformation requires NO ADDITIONAL energy or information
(SELF ASSEMBLY)
Protein folding
Amino acid sequence contains all information necessary for folding into a specific three-dimensional structure
Protein Folding
Proteins, in general, do NOT fold as they are synthesized on the ribosome
Folding of RNAse A in the test tubeFolding of RNAse A in the test tube
denaturation renaturation
Incubate proteinin guanidine
hydrochloride(GuHCl)or urea
100-folddilution of proteininto physiological
buffer
Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181, 223-230.
- the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation
Thus: “protein folding is a spontaneous process that does not require the assistance of extraneous factors”
Protein Folding
Many proteins fold by Assisted Self Assembly
Correct assembly (native conformation) requires assistance
by CHAPERONES
Protein unfolding = DenaturationLoss of structure and function
– Heat– Extreme pH– Detergents– Urea
Protein unfolding = DenaturationWhy do these conditions cause loss of
structure and function?– Heat– Extreme pH– Detergents– Urea
Lysozyme
Lysozyme
Tertiary: complete three-dimensional
structure
Quaternary: arrangement of subunits
(in multisubunit protein)
Hemoglobin
Quaternary structure
• Held together by weak interactions between side (R/functional) groups as well as covalent disulfide bonds
Structure-function relationship• Function is derived from structure
• Structure is derived from sequence
Sickle-cell diseaseNormal red blood cells Sickle shaped red blood cells
Due to single amino acid change in haemoglobin
Sickle-cell disease
Sickle-cell disease
• Single specific amino acid change causes change in protein structure and solubility
• Results in change in cell shape
• Causes cells to clog blood vessels
Amino acids