GCD 3033 - Chapter 4

What does the function of a protein depend on?

The 3D structure

What do enzymes do?

Catalyze reactions

What do structural proteins do?

Support and organize compartments

What do transport proteins do?

Carry small molecules from one compartment to another

What do motor proteins do?

Move larger components within cells or execute cellular movements

What do storage proteins do?

Store molecules needed for growth and development

What do signaling proteins do?

Mediate communication within and between cells

What do receptor proteins do?

Receive signals and transmit them to intracellular targets

What do gene regulatory proteins do?

Bind DNA and turn genes on and off

What do proteins do?

Execute most cell functions

Polypeptide

A long chain of amino acids linked together by covalent peptide bonds

How many amino acids are there?

20

How do amino acids differ?

Structure of the side chains

What kind of non-covalent bonds influence protein folding?

hydrogen bonds, electrostatic interactions, van der Waals forces

How strong are non-covalent bonds?

Weak

How do hydrophobic interactions non-polar side chains?

Side chains tend to cluster together to minimize their interactions with the H₂0 molecules present in the aqueous environment

Where are polar side chains found?

On the outside surface, where they can interact with water and other molecules (hydrophilic).

What happens to polar amino acids within a folded protein?

Form hydrogen bonds with other polar amino acids or the polypeptide backbone

What does the final folded structure rely on?

The lowest free energy state

How are proteins unfolded?

Solvents that disrupt non-covalent interactions

How is protein folded assisted?

Molecular chaperone proteins

What kind of structure is the alpha helix and beta sheet?

Secondary structure

What bonds form alpha helixes and beta sheets?

hydrogen bonds between N-H and C=O groups in the polypeptide backbone

How many turns per amino acids are in an alpha helix?

3.6

How many hydrophobic amino acids are in alpha helices that span a membrane lipid bilayer?

20

How is the beta sheet formed?

Hydrogen bonds between the peptide backbone in different polypeptide strands

How do you know when the polypeptide strands of a beta sheet are parallel?

When the strands run in the same direction (N to C terminal)

How do you know when the polypeptide strands of a beta sheet are antiparallel?

When the strands run in opposite directions

How do protein aggregates form?

When proteins fold incorrectly

What kind of diseases are linked with the formation of protein aggregates?

Neurodegenerative disorders

What are prion diseases?

A neurodegenerative disease caused by infectious proteins that stimulate protein misfolding

What are protein domains?

Small stretches of amino acids that can fold independently into a stable structure

How many amino acids are in a protein domain?

100-250

What is the tertiary structure of a protein?

Full three-dimensional structure of a polypeptide chain, with its multiple secondary structures and domains

What is the quaternary structure of a protein?

Multiple polypeptide structures

What are protein families?

Groups of proteins which share both sequence and structural similarities, but perform slightly different functions.

What are serine proteases?

A family of proteins that can cleave peptide bonds in a polypeptide chain.

What is a binding site?

A region on a protein surface that interacts with another molecule through the formation of non-covalent bonds

What are globular proteins?

Large and complicated proteins that fold into compact shapes with an irregular surface

What are fibrous proteins?

Proteins with an elongated 3D shape

Where are fibrous proteins usually found?

Extracellular matrix

How is collagen structured?

Three polypeptides that form a triple helix

What are bonded collagen molecules called?

Collagen fibris

What is the structure of elastin?

Polypeptides cross-linked in a rubberlike elastic meshwork

What group uses disulfide bonds (S-S)?

Cysteines

Ligands

Molecules that proteins bind to

How do proteins bind with ligands?

The formation of many weak non-covalent bonds and hydrophobic interactions. The strength and specificity of the binding depends on the number of these interactions.

Binding site

The region of a protein that interacts with the ligand

What kind of proteins bind ligands with high specificity?

Antibodies (IgG)

What is the structure of IgGs?

Two large or heavy chains and two smaller or light chains held together by disulfide bonds

Constant regions

Areas in the polypeptide sequence which are conserved between different antibodies

How is the antigen-binding site formed?

Small variable loops in the heavy and light chains. Changing the length and sequence of the variable loops will alter their binding specificities for different antigens without affecting the structure of the rest of the antibody molecule.

What do enzymes do?

Bind their ligands (known as substrates) and convert them into chemically modified forms (by making and breaking covalent bonds).  They speed up chemical reactions by acting as catalysts

How do enzymes lower activation energies to speed up reactions?

They can bring two substrates together in the proper orientation, they can rearrange electrons in a substrate to favor a reaction, and/or they can strain the conformation of a substrate to favor a reaction. They can bring two substrates together in the proper orientation, they can rearrange electrons in a substrate to favor a reaction, and/or they can strain the conformation of a substrate to favor a reaction.

How do some drugs work?

Blocking the activity of a certain enzyme

Allosteric regulation

When a regulatory molecule binds to a secondary site on a protein to inactivate or stimulate a protein

What is an example of allosteric regulation?

Feedback inhibition

How is protein phosphorylation and dephosphorylation used to control protein activity?

Phosphate groups contain two negative charges and their addition to specific serine, threonine, or tyrosine side chains can significantly alter protein conformation

How is phosphate addition catalyzed?

With a protein kinase using ATP

How are phosphates removed from amino acid side chains?

Protein phosphatase

What does the addition of palmitate to cysteines do?

Drives proteins to cell membranes

What does the attachment of ubiquitin do?

Targets a protein for degradation

Can protein phosphorylation be used to control assembly of larger protein complexes?

Yes

GTP binding proteins

molecular switches that are regulated by the cyclic gain and loss of a phosphate group

What conformation are GTP binding proteins in with bound GTP

Active

What conformation are GTP binding proteins in after hydrolyzing GTP to GDP?

Inactive

How do motor proteins move in one direction?

Energy of ATP binding, hydrolysis, and release to drive the conformational changes needed

What are scaffold proteins?

Proteins that contain binding sites recognized by multiple proteins. Allows the formation of a protein complex.

Biomolecular condensates

Proteins organized into discrete regions of cytoplasm

How can you isolate a protein of a particular cell type?

Break open the cell (poke holes in the cell membrane) to make a cell homogenate or extract that contains all the cell organelles in a thick soup of cytoplasm.

What methods can you use to break open a cell to make a cell homogenate or extract?

1. sonication (high frequency sound)

2. mild detergent treatment

3. forcing cells through small holes under high pressure 

4. mechanical shear

Differential centrifugation

Using centrifugal force to separate cell components by size. Larger components fall to the bottom first.

What is velocity sedimentation used for?

To separate larger protein complexes based on the rates that they move through a shallow gradient of sucrose.  Larger, denser complexes will move faster than smaller, less dense complexes, but one must optimize the time and speed of centrifugation empirically.  If you spin too long, everything will end up at the bottom of the centrifuge tube.

What is equilibrium centrifugation used for?

 To separate components based on their buoyant density, independent of size and shape. The sample is applied to a steep gradient of sucrose or cesium chloride and centrifuged until it reaches the position where its density matches that of the surrounding solution. You need to spin long enough for the sample to reach equilibrium, but once it does, it will not change its position. It is often used to isolate nucleic acids (DNA, RNA) or membrane fractions of different buoyant density.

What aspects can column chromatography be used to separate proteins by?

Size, charge, affinity

Ion-exchange chromotography

Separating proteins based on relative charge

Gel filtration chromatography

Separates proteins according to their size. The beads used in gel filtration are porous. Larger proteins cannot enter the pores and travel around the beads and elute first. Smaller proteins can enter the pores and so experience a larger effective volume. They elute more slowly from the column. Beads are available in a number of different pore sizes, and so it is possible to design columns to separate a wide range of protein sizes.

Affinity columns

Contain a matrix that is covalently bound to a molecule that interacts specifically with the protein of interest. This molecule may resemble an enzyme’s substrate, or it might be a specific antibody that recognizes an antigen on the protein surface. Other proteins will wash through the column, but the protein of interest binds to the beads. It can later be eluted from the beads by washing the beads with an excess of substrate, or by adjusting the pH or salt concentration of the buffer.

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

A way to measure relative molecular weight of protein complexes

Immunoaffinity chromatography

When antibodies are bound to beads to purify a protein of interest from a mixture of molecules

How many different amino acids are used in making proteins?

20

Which parts of amino acids are involved in peptide bonds?

The amino group on one amino acid and the carboxyl group on the other

Which part of an amino acid gives it its unique properties?

The side chain

In a folded protein, the nonpolar (hydrophobic) amino acids tend to be:

tucked away inside the protein.

What provides the information necessary to specify the three-dimensional shape of a protein?

The protein’s amino acid sequence

The biological activity of a protein is determined by its:

amino acid sequence.

A protein can be unfolded by a process called:

denaturation.

Which hydrogen bonds have been found to stabilize a polypeptide’s folded shape?

All of the above

What does the term protein domain refer to?

A segment of a protein that can fold independently into its own compact, three-dimensional structure.

Why are α helices and β sheets common folding patterns in polypeptides?

Amino acid side chains are not involved in forming the hydrogen bonds, allowing many different sequences to adopt these folding patterns.

Those portions of a transmembrane protein that cross the lipid bilayer usually consist of which structures?

An α helix with mostly nonpolar side chains

What does the primary structure of a protein refer to?

The amino acid sequence of the protein

What is the name for a modular unit from which many larger proteins are made?

Protein domain

What are protein families?

Evolutionarily related proteins that are similar in amino acid sequence and three-dimensional conformation.

What is the definition of a binding site on a protein?

Any region that interacts with another molecule through sets of noncovalent bonds.

The most common covalent cross-links in proteins are sulfur–sulfur bonds that form between two amino acids with —SH (thiol) groups as side chains. Which amino acid has this side chain?

Cysteine

The ability of a protein to bind selectively and with high affinity to specific molecules is due to which types of bonds?

Weak, noncovalent bonds

How many identical binding sites exist on an antibody?

2

What determines the specificity of an antibody for its antigen?

The amino acid loops in its variable domain

Which of the following is NOT true?

Enzymes require an input of energy from ATP for activation.