Flashcards in Protein Structure Deck (51):
Approximately 9,000 people/year
Many cancers have an abnormal form of the protein Ras
It is permanently bound to GTP
Continuous cell division
Inhibiting Ras stops cell division
Protein Kinase Cascade
Begins with a growth factor like a hormone, binds to receptor protein, which changes its conformation (new chemical interaction) causes receptor to autophosphoylate.
Then activation cascade in cytosol of cell, eventually final.
Signal in cascade goes into nucleus and turns in transcription and translation
Representations of ras structure
Ca backbone trace, ball and stick, ribbon model, solvent-accessible surface(shows regions of reactivity and polarity
Made up of amino acids
Peptide bonds join amino acids
• Amino acids (aa) are the monomeric building blocks of proteins
There are 20 different aa. (22 if you consider selenocysteine and pyrrolysine)
All organisms on earth havethe same 20 aa.
Each protein being synthesized is made from the different arrangements of the aa.
Amino acid structure
Net charge is zero (zwitterion)
Four things attatched to carbon- H, R, carboxyl group and amide group
Stereoisomers are formed
Peptide bond formed between carboxyl and amino groups
Amino Acids react differently with water
Amino acids are also broken down into groups of those with side-chains that are:
acidic or basic
R group determines hydrophillicity
Hydrophilic amino acids
Hydrophilic amino acids dissolve easily in water. Acidic aa readily donate a proton and basic aa take up a proton
Often find histidine in the active site
Hydrophilic basic AA
Side chain has a positive charge after accepting a H+ under physiological condition
Under physiologic conditions polar amino acids are uncharged, but are able to form H bonds
Side chains tend to have partial (+)or (-) charge able to form H-bonds with other amino acids and associate with water.
Sulfur in cysteine links proteins together
Asparagnine and glutamine good acceptors/donors of H, cannot be phosphorylated
Non polar amino acids
These side chains often point to the inside of the protein where they form hydrophobic associations with each other, notice the methyl groups
Methionine important for protein synthesis- sulfur in R group
Phenylalanine and tryptophan- aromatic
Interesting Amino Acids
Cysteine- forms covalent disulfide bonds, Rarely see disulfide bridges in the cell because the cytosol is a reducing environment
Glycine- smallest r group, see it in stems and loops of proteins, not a stereocenter
Proline- cyclic, forms bends or kinks, disrupts structure
• Polypeptides are linear polymers of amino acids.
• Peptides usually contain less than about 20-30amino acids.
• Proteins usually contain 100 or more aa
How are polypeptide bonds formed?
Electrons shared between carbonyl group and peptide (carboxyl and amino group)
structure of peptide unit
The peptide unit is planar because the carbon-nitrogen bond has partial double-bond character due to resonance. Electrons being shared are in the sp2 orbital
R-groups protrude away from the amide plane of the peptide bond- proteins can't simply fold, need some space in between
Levels of structure in proteins
Primary- order of amino acids
Secondary- alpha helices, beta pleated sheets, random coils, turns
Tertiary- Sum of all the secondary structures, stable 3D structure. Domains and motifs
Quaternary- number of polypeptides, multiple proteins brought together, acting as subunits of multiple proteins
Structure and function
• The 3D shape of protein is determined by its amino acid sequence
• The unlimited 3D shapes (conformation) that proteins can assume allows them to perform a wide range of functions.
• Proteins also have directionality- functionality and reactivity
Functions: regulation, structure, movement, catalysis, transport, signaling
Sequence of amino acids, determined by sequence of nucleotides, play a primary role in determining all other levels of structure.
Side chains on amino acids effect folding of the polypeptide into stable secondary structures
Interactions between the amino acid side chains play a major role in determining the stability of the tertiary structure.
In general, the lowest energy state is attained byfolding hydrophobic aa inside the protein and hydrophilic aa outside
How is primary structure determined?
Sense strand- coding or non template
Antisense strand- non coding or template -> RNA -> proteins. Message AA in triplets to code for proteins (codons)
DNA has to be denatured so that there are single base pairs available
Methionine is the start codon, but is cleaved during/after translation
Genetic code degeneracy
multiple codons for amino acids. Wobble base pairs
Comparison of many protein 3D structures revealed 2 common repeating units: alpha-helix and beta-sheet.
Both structures are stabilized by H-bonds between the peptide bonds in the backbone
Since side chains are less important, many sequences can assume these same repeating structures
Frequently represented by a cylinder
• Right handed helix
• 3.6 residues/turn
• H-bond between every 4th aa
• Forms a rigid structure
• R groups project out from helix so they are available to react
• H-bonds are parallel to axis of the helix
Alpha helix of a transmembrane protein
More nonpolar amino acids in transmembrane protein. A proteins neighbors determine the hydrophobicity. How hydrophobic each is depends in neighbors
If you know the primary structure of a protein can you determine if there is a transmembrane region?
On a hydrophobicity chart of the residue there will be a peak at the transmembrane region
Frequently represented by arrows
-Planar peptide bonds withbend at alpha C
-H-bonding between one beta strand and another
-R groups alternate above and below sheet
-They can be parallel or antiparallel
Often see proline here because ring form doesn’t fit into helices or sheets,and there aren’t sites available for H bonding.
• U-shaped or unshaped structure
• 3-4 residues per unit
• Usually formed by glycine or proline
• Turns are needed to connect different helices/strands
• Are often located on the surface of a protein
• Longer turns are called loops and random coils
Motifs of Protein Secondary Structure
Zinc finger motif
(Both these DNA binding regulatory proteins)
Coiled coil motif- common in collagen, transcription factor
Changes to Primary Structure
• Cascade effect to other levels of protein structure
• Glutamate to valine- Equivalent of a water repellant sticky-patch on HbS, hemoglobin can't bind oxygen, sickle cell anemia
Globular formation after all of the local structures are packed into place
Domains of the protein, folds into appropriate confirmation in absence of the rest of the protein
Can refer to them by structure or function. Typically formed by a continuous (~40) amino acid sequence.
Ex: 1. DNA-binding domain leucine zipper
2. Transcription activation domain
Domain may contain one or more motifs- Motif part of the domain. Region of functionality that binds DNA (secondary structures)
Categories of tertiary structure
Globular, fibrous, membrane
Sub-unit structure of a protein
There are interactions that hold the sub-units together- most important are non covalent bonds
Each polypeptide of a multimeric unit is called a subunit
Homotrimer- three subunits, all the same. Hetero/homo, dimer/trimer/tetramer
• Multiple proteins associated with one another and are usually involved in a related series of reactions
• Important for transport of products
• Are not considered highly stable associations, but in closecontact can be stabilized by non-covalent bonding
Different proteins with own role, have to be associated together for a mechanism to begin. Don't form one protein like quaternary structure
Proteins (along with other macromolecules) can associate into larger structures called macromolecules assemblies
Subunits (sugars, amino acids, nucleotides) form macromolecules (with covalent bonds). Macromolecules (globular proteins, RNA) form macromolecular assemblies (ribosome) with non covalent bonds
Why do cells need protein complexes (i.e. transcription initiation machine)?
Different cominations of proteins do different thing. Adding different proteins modifies the function. Each one of these proteins is a way in which is function can be regulated. Wy of controlling what macromolecular assembly does in the cell
• The complexity likely evolved gradually through evolution, in which different subunits were retained if it benefited fitness.
•Different subunits can be regulated individually, such that the activity of the complex would respond to different signals.
A special class of proteins: Enzymes
• Terminology - name indicates its structure or function.
• Ends in the suffix -ase, protease, ribonuclease
• Enzymes are highly specific- don't want unnecessary reactions, but a few aren't specific
• Function by lowering the activation energy of the reaction
• The active site of an enzyme frequently fits the transition state better than the reactants or products
• Catalyze reactions by converting substrates to reactants without altering the enzyme
• Increase the overall rate of reaction but not the free energy
Temperature and reactions
temperature can perform the same function as a catalyst- speed up reactions
But sometimes temperature can do things out I don't want- can denature the protein (egg whites are denatured), but some proteins can renature
Free energy of a reaction
• Measure of potential energy
• Gibbs stated that “all systems change in such a way that free energy is minimized”
• ∆G = Gproducts - Greactants
The free energy of the products will be lower than the reactants if the reaction is exergonic (∆G <0)
Breaking bonds of reactants releases potential energy
Model of enzyme action
Input of energy is required to initiate reactions. ∆G only determines the direction, not the rate of reaction
Enzyme is most complementary to transition state. Enzyme stabilizes this state, lowers the activation energy
1) initiation-reactants bind to the active site in a specific orientatio
2) transition state facilitation- interactions between enzyme and substrate lower the Ea
3) termination- products have lower affinity for active site and are released. Enzyme unchanged
• An unstable intermediate, but the intermediate with the highest potential energy
– This activated state requires energy to cause a chemical bond to be strained or for electrons to be excited in the molecule. Enzyme active site bends around it and strains the bonds, gives them more kinetic energy and makes the reaction go faster
– Some transition states require covalent bonding of the substrate to the enzyme
How do enzymes catalyze reactions?
Change reactivity of substrate (substrate can acquire a charged region because of the enzyme)
When the substrate binds to the enzyme's active site, the enzyme changes shape slightly, this induced fit results in a tighter binding of the substrate to the active site
Always hints moving in and out of the active site
What actually happens in the active site
R groups in the active site stabilize the transition state of a substrate Ex:RNA
1) active site is empty
2)substrate (RNA) fits into active site. Proton from substrate transferred to R-group 1 of active site
3) transition state is stabilized by R-group 2, proton from R group 3 is transferred to substrate, splitting it in 2
Limits to catalysis
Substrate concentration- At some point there is a maximum speed of reaction, at some concentration it will level off no matter the substrate level. Limited by number or enzymes available to catalyze the reaction, saturated
Different from uncatalyzed reactions
Breakdown of alcohol
Ethanol is broken down in two steps- usually one fast and one slow enzyme.
Converted to acetaldehyde, and then acetic acid
Two different versions of the enzyme for the second reaction
Single aa change in fast-acting at active site
Effects of physical conditions
Enzymes from different organisms may function best at different temperatures
Enzymes from different organisms may function best at different pHs
Not all reactions are “spontaneous”
Products have higher energy than reactants- endergonic
Many endergonic reactions are “coupled” with exergonic reactions
Some reactions are coupled to the the breaking of theterminal phosphate bond of ATP
Coupling ex: exergonic reaction (loss of Pi from ATP) + endergonic reaction (B gains Pi from ATP) = endergonic a reaction (B loses Pi)- energy is required to add a monomer to a polymer
How are enzymes regulated?
• Feedback Inhibition
• Allosteric Interactions
• Enzyme modifications
• Enzyme levels
In synthesizing a molecule there are many steps, each using a different enzyme.
Feedback inhibition occurs whenthe final product binds to a site on the first enzyme.
This changes the conformation ofthe enzyme and it’s no longer active.
Ex: cellular respiration- the first three reactions require energy, but there is control at the committed step of glycolysis. ATP inhibits Phosphofructokinase, Doesn't need to waste energy, unbinds when ATP level drops
The conformation of the enzyme will change shape if it binds with a regulatory molecule (called a ligand), this molecule is called an allosteric effector. It binds somewhere other than the active site
Competitive inhibition prevents the Substrate from binding to the enzyme by binding to the active site, allosteric regulation changes the enzymes shape to activate or inactivate it
Ex: Calmodulin- 150 aa with 4 Ca++ binding sites, Typical cell has > 106 molecules. Can bind various proteins and turnthem on or off- Smooth muscle doesn't have troponin. Calcium released binds calmodulin, Calmodulin binds myosin light chain kinase which initiates contraction and activates myosin ATPase that is responsible for muscle contraction. Inorganic and organic cofactors
Regulation via Protein Modification
Kinase adds a phosphate group, Phosphatase removes a phosphate group
Adding or removing a phosphate can activate or inactivate a protein
Phosphorylation can change conformation ligand binding site binding with another protein
Protein kinase cascade