The 3D Structure of Proteins
Chapter 4
Outline
• the levels of protein structure - 1⁰, 2 ⁰, 3 ⁰ and 4 ⁰
• protein folding
• as we discussed last week, proteins are chains of amino acids linked together (covalently!) by peptide bonds
• proteins can – and very often do – __________ up upon themselves creating distinct three-dimensional shapes
- we call this a protein’s _____________________________
• as you can imagine, there are many different possible ways to fold up an amino acid chain (almost infinite)
• but only one of these forms is correct and therefore functional
- this is the protein’s ________________ CONFORMATION • when it comes to proteins…
- STRUCTURE = FUNCTION
STRUCTURE = FUNCTION
Proteins are exactly the same (conceptually)
- the shape of the protein lets it do the job that it does…
The Levels of Protein Structure – Primary (1⁰)
• so, proteins can fold up into many different shapes, but only one of those shapes is the RIGHT
SHAPE that allows the proteins to do its cellular job – again, this is the ____________ CONFORMATION
• let’s fold a protein….
• the PRIMARY STRUCTURE of a protein is merely the _____________________ of amino acids in its polypeptide chain
• that’s easy… just remember that ________________________ matters - we always go N → C (G-P-F-M is different from M-F-P-G)
• the primary structure is a one-dimensional start to the three dimensional final protein structure
• proteins fold into the correct shape because of their particular amino acid sequence – therefore, SEQUENCE = STRUCTURE = FUNCTION
The Levels of Protein Structure – Secondary (2⁰)
• the book says that secondary structure is “the hydrogen-bonded arrangement of the backbone of the protein”
- that’s correct, but a bit much…
• I like to say that secondary structures are “regions of ____________________ order in a larger ordered protein”
• the 3rd floor is certainly part of the entire building and the building would not be complete
without that 3rd floor
• in much the same way, secondary structures are part of the larger entire protein, and the protein would not be complete without them, but secondary structures are ordered in and of themselves
• we have three covalent bonds when we’re discussing amino acid residues
- the ________________ bond is static – no free rotation due to _______________________
- the other two bonds are true ______________ bonds and therefore free to rotate (these are the bonds holding the α carbon to the amino and carboxyl groups)
• this allows quite a bit of flexibility
• the two most common types of secondary structures are α helices (plural of helix) and β sheets • these are repeating structures of local order (hence, 2⁰ structure)
- they are entirely dependent on ___________________ α helix
• the α helix is a ___________ (spiral staircase) with a narrow, long shape
• it is stabilized by H-bonds that are _________________ to the helix and between amino and carboxyl groups of the peptide backbone
• a carboxyl group is H-bonded to the amino group of the amino acid residue ________ away in the chain
• proteins can contain no α helices, can be almost 100% α helices and everything in-between • PROLINE disrupts α helices
- its weirdo shape (ring that includes the amino group) does not fit into the repeating pattern of a helix
- there is no free ________________ between the α carbon and amino group nitrogen in proline - there is no H-bonding from the amino group of proline
• side chains can also disrupt an α helix
- too many side chains of the same _______________ in a row
- too many large ________________ side chains in a row
• in α helices the side chains project out from the helix itself, so they’re free to get in each other’s way
β sheet
• the peptide backbone in the β sheet is almost completely _____________________ - stretched out
• this stretched out form can double back on itself or different backbones from different peptides can line up next to each other
- either way, _________________ hold them together - these are intrachain or interchain H-bonds, respectively
• if the peptide chains run in the same direction as each other (N→C) a ____________________ β sheet is formed
• if the peptide chains run in opposite directions as each other (N→C; C←N) an ______________ β sheet is formed
• β sheets are often called pleated sheets because they pinch or pucker a bit due to the H-bonds pulling on each β strand
• the side chains are perpendicular to the peptide bonds in the other direction
Turns, Motifs and Domains
• again, these secondary structures are great, but useless unless they can give us a three dimensional shape that folds in on itself
• this requires some pretty sharp turns
• __________________ is usually seen in sharp turns - why…?
• __________________ is also often seen in turns
• _______________ are small clusters of secondary structures that are commonly found together – they sometimes do a conserved function
• ________________ are usually larger regions of proteins
- the defining characteristic of a domain is that it is _________________ - it can do its job outside of the context of the larger protein
• the “helix-turn-helix” motif cannot bind DNA outside of the larger protein • the “Gal4 DNA binding domain” can bind DNA all by itself
- domains _____________ function
Protein Conformations: Fibrous vs. Globular
• fibrous proteins tend to be long, narrow, extremely strong and stable
- they are also _________________ (they don’t DO anything)
- and therefore, they are _____________________
• globular proteins fold back on themselves often and create a shape that’s roughly __________ - residues from far away in the amino acid chain can be brought very close
- allows catalytic sites to form (these are our machines)
The Levels of Protein Structure – Tertiary (3⁰)
• tertiary structure is the three dimensional arrangement of all the atoms of a single polypeptide/protein chain
• tertiary structure includes the arrangement and positions of all the side chains, _____________ groups (noncovalently bound cofactors that help a protein do its job), and the arrangement of the secondary structures with respect to each other
• said simply, the tertiary structure is the completely __________________ protein (but only for a single polypeptide chain of amino acids)
• folding can bring amino acids that are far apart in the chain very close together and allow them to interact
- the side chains of these amino acids can then interact in a favorable way • a folded protein is a lot like a pile of string
• most of these forces are non-covalent forces
- bonds: between polar groups in the _______________________________ (remember, H-bonds between amino and carboxyl groups of the backbone contribute to secondary structure)
- ______________________ interactions: favorable interactions between oppositely charged side chains (acidic and basic groups)
- where would we find these side chains?
- hydrophobic interactions: perceived interactions between __________________ side chains - why perceived?
- where would we find these side chains?
• covalent forces also contribute to the overall shape of a protein
- of course, the peptide bonds of the amino acid chain are essential to the integrity of the protein
- what other covalent bond have we discussed in the context of amino acids?
- __________________ bonds can hold ____________________ together as long as they are close to each other in physical space
• it’s important to note that not every single protein using all of these forces
- but you’re likely to find H-bonds, _______________________ interactions and hydrophobic interactions in the vast majority of proteins
• the sum total of forces stabilizing a protein give it its conformation
The Levels of Protein Structure – Quaternary (4⁰)
• so, after tertiary structure, our protein is completely folded into its native conformation and functional
• all done, right…? - not always
• some protein machines are actually made up of more than one single polypeptide chain of amino acids
• these machines are called COMPLEXES and each individual polypeptide chain is called a SUBUNIT
- so, two or more subunits make up a complex
• subunits of complexes may be identical to each other or different
• common complexes are dimers, _____________, and _______________ (after that its
_______________ for all the rest)
• subunits interact with each other through noncovalent forces - i.e., H-bonds, hydrophobic interactions, van der Waals, etc. - or through disulfide bridges (covalent)
• because these subunits are so intimately in contact, a change to one subunit can be
‘______________________’ to another
- if someone fainted (and I’m sure someone did), the people around the faintee step back to give him/her room
- their stepping back jostles and crowds the people around them who in turn step back and jostle and crowd others
- next thing is you get jostled and crowded and you’re like ~50 yards away from the faintee… • the same thing happens with many proteins (but not all)
- we call this property ______________________ (as in, an ____________________ interaction)
Protein Folding
• in many ways, protein folding deserves its own course - we’ll cover it in the three minutes I have left…
• as we’ve already said, the amino acid sequence (primary structure) directly determines the overall shape of a folded protein (tertiary)
• but even with that direct relationship, we’re still not very good at predicting the 3D shape of a folded protein given only its amino acid sequence (… despite what the textbook says…)
• the largest contributor to protein folding is ______________________________
- the nonpolar side chains seek refuge from water inside the globular protein, while the polar side chains remain on the exterior to interact favorably with water
• remember, the hydrophobic side chains don’t really like each other - it’s just that water is their common enemy
• correct protein folding is critical to the functioning of the protein and the ________________ of the organism
- Mad Cow, Alzheimer’s, Parkinson’s, and Huntington’s diseases are all due to proteins improperly folded, exposing hydrophobic residues on their exteriors and then
_____________________ (clumping up)
• as much as the information for proper folding is contained in the primary amino acid sequence, proteins don’t always fold up properly on their own
- you know it’s not good to cut class because you’d like another half hour in bed, but you still do it…
• specialized proteins called ________________________ keep an eye on this and make sure individual proteins fold into their native conformation
• proteins can be unfolded or DENATURED using anything that disrupts the noncovalent forces holding them in their 3D shape:
- heat (remember, these forces are weak – heat can break them)
- extremes of ______ (these disrupt electrostatic interactions by protonating or deprotonating charged side chains)
- treatment with detergents (detergents are ______________________ and so interact favorably with the nonpolar side chains, drawing them to the exterior of the protein; this literally turns the protein inside-out)
• for complete denaturation, we also use a ____________________ agent - DTT, BME, etc.
Determining Protein Structure – in Brief
• the three dimensional shape of any given protein can be determined (visualized) in two ways: - X-ray crystallography
- NMR (nuclear magnetic resonance)
• we do not have time to go into how these techniques work, but you are responsible for knowing that they exist
• for X-ray crystallography, perfect crystals of pure proteins are made where all the proteins in
the crystal are _____________________ the same way - x-rays are then shined through the crystal
- how the x-rays are disrupted/diffracted gives information regarding the overall shape of the individual proteins
• NMR uses the same technology as MRI
- a large _______________ makes hydrogens vibrate
- locating these protons allows us to infer the positions of all atoms
Summary
• the one 3D folded shape a protein can take that allows it to be functional is called its ________ CONFORMATION
• STRUCTURE = FUNCTION
• primary structure (amino acid sequence) • secondary structure (α helix, β sheet, turns) • tertiary structure (fully folded polypeptide chain) • quaternary structure (complexes)
• forces holding proteins together
• determining protein structure (x-ray crystallography vs. NMR)
