Over the next 2 lectures, our goal is to - understand the chemistry of proteins and protein folding - recognize different

structural levels of proteins and the role of structure on protein function

- recognize and analyze the relationship between

similar proteins in terms of protein domains and

protein families
- understand how protein function is regulated

1. Protein composition Amino acids 2. Protein shape and structure Bonding other small molecules 3. How proteins work: binding to other molecules Protein-Protein interactions Protein-Small Molecule interactions 4. Protein domains and Protein families

5. How protein function is controlled A. Multiple binding sites B. Phosphorylation C. Nucleotide binding and hydrolysis D. Proteolytic cleavage

Water is an important component in all cells, along with other stuff:

We are organic beings: carbon-based. Macromolecules are synthesized from the appropriate precursors. Link monomers to generate polymers.

Table 4-2 Essential Cell Biology ( Garland Science 2010)

What do proteins do?

Enzymatic reactions - covalent bond formation and breakage Structural proteins - support cells and tissues Transport proteins - carry small molecules, ions Motor proteins - generate movement in cells and tissues Storage proteins - store small molecules and ions Signal proteins - carry signals from one cell to another Receptor proteins - detect and send signals to cell response machinery Gene regulatory proteins - interact with DNA and control gene expression Special-purpose proteins - variable (glow, antifreeze, attachment, etc)

How do they do all those things???

Protein Structure = Protein Function

Amino Acids:

One rendition (Alanine):

Another rendition:

Figure 4-1 Essential Cell Biology ( Garland Science 2010)

Figure 2-22 Essential Cell Biology ( Garland Science 2010)

2. Shape and Structure: Polypeptide chain = 1o structure

Figure 4-2 Essential Cell Biology ( Garland Science 2010)

Figure 4-5 Essential Cell Biology ( Garland Science 2010)

Multiple interactions between AAs and peptide backbone:

Figure 4-6 Essential Cell Biology ( Garland Science 2010)

Common 2o structure: -helix

Hydrogen bonding of backbone N-H to C=O, 4 peptide bonds apart

Figure 4-10ac Essential Cell Biology ( Garland Science 2010)

 helices are common in proteins that cross membranes (transmembrane proteins)

The -helices may interact to form coiled-coils (ie keratin, DNA transcription factors)

Figure 4-12 , 13 Essential Cell Biology ( Garland Science 2010)

2o Structure: -pleated sheet

AAs above, below plane

Figure 4-10df Essential Cell Biology ( Garland Science 2010)

Anti-parallel or parallel structure

Confer rigidity, strength (silk, biological antifreeze)

Figure 4-14, 15 Essential Cell Biology ( Garland Science 2010)

Chemical nature of the amino acids will cause interactions between them, non-covalent bonds. Contribute to structure.

Figure 4-4 Essential Cell Biology ( Garland Science 2010)

3. 3o structure: a three dimensional globular polypeptide chain

Figure 4-17 Essential Cell Biology ( Garland Science 2010)

Figure 2-31 Essential Cell Biology ( Garland Science 2010)

Covalent disulfide bonds contribute to stabilizing protein structure:

Figure 4-26 Essential Cell Biology ( Garland Science 2010)

Figure 4-25b Essential Cell Biology ( Garland Science 2010)

Cysteine: Bovine insulin:

S S G-I-V-E-Q-C-C-A-S-V-C-S-L-Y-Q-L-E-N-Y-C-N S S

SH
CH2 H2N-C-COOH H

S S F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-L-A

Presence of Proline in a structure can make a kink in the polypeptide chain. S-E-G-G-A-L- P N Q V

Proline: CH2
CH2 CH2 H2N-C-COOH H

Homodimer

4o structure: more than one polypeptide chain interacting in complex

04_22_protein subunit.jpg
Homotetramer Hemoglobin: 4 subunits 2 chains 2 chains

Macromolecular interaction through various bonds:

The stronger the bonds, the more stable the interaction.

Protein structure can be denatured, and may reanneal to regain function.

04_07_Denatured prot.jpg

Not all denaturing agents can be reversed - HEAT. If you alter STRUCTURE, you can alter FUNCTION.

Binding sites (or pockets) within a protein:

Figure 4-28 Essential Cell Biology ( Garland Science 2010)

3. How Proteins Work: interact with other proteins and/or small molecules in very specific fashion. Interaction determined by structure! Thing that binds

Region of binding

04_30_selective binding.jpg

4.27

Some proteins require small molecule binding to be functional:

Hemoglobin (Fe2+) Other examples: Botulinum toxin (protease) binds Zinc Transcription factors (DNA binding) bind Zinc Amylase (digestive enzyme) binds Cl-

Without associated factor, protein not functional.

Protein subunits (polypeptide chains) can assemble into filaments sheets, or spheres. Self-organizing structures when proper elements present.

Figure 4-21 Essential Cell Biology ( Garland Science 2010)

An example of self-assembly gone awry

Mathews and van Holde, Biochemistry

4. Protein domains and Protein families Proteins with similar overall structure may have similar function can be grouped as a protein family.

Elastase and chymotrypsin are family members of a group of enzymes that digest other proteins (proteases).

Proteins can have regions of amino acid sequence that have a particular structure/function within the protein as a whole. These microfunction regions are protein domains.
For example:

Phosphorylated Tyrosine Binding PTB Domain SH2 Domain Phospholipid Binding BAR Domain BEACH Domain C1 Domain GLUE Domain GRAM Domain PH Domain Protein Degradation F-Box Domain HECT Domain RING Domain Proline-Rich Sequence Binding EVH1 Domain GYF Domain SH3 Domain Ubiquitin Binding CUE Domain GAT Domain MIU Domain Vesicle Trafficking EH Domain SNARE Domain

C2 Domain PX Domain
SOCS Domain

WW Domain
NZF Domain

Example of a Protein Domain: SH2 domains

Src-homology 2 (SH2) domains are modules of approximately 100 amino acids that bind to specific phosphotyrosine (pY)-containing peptide motifs.

http://www.cellsignal.com

Src SH2 domain bound to phosphotyrosine peptide

While they have similar structure/function, their different specificities are due to the differences in their amino acid sequences in the site that cleaves the target proteins.

These members of the protease enzyme family could have similar structures, similar functions, and similar protein domain composition, but have unique differences related to the specificity of their function.

How are these differences generated? Mutation and selection

GONE: UONE GONH NONE GCNE GONI BONE GNNE GOLE GKNE GOVB GFNE GOWE GONJ GFNE GJNE GOIE GPNE OONE GOZE XONE DONE GGNE GENE GYNE

Example: evolution of resistance of malarial parasite to drug pyrimethamine In the original DHFR protein before the drug was widely used: Amino acid 108 = Serine (S) Amino acid 59 = Cysteine ( C) Amino acid 51 = Asparagine (N) Amino acid 164 = Isoleucine (I) First, then, then, finally, Amino acid 108 = Serine (S) was replaced with asparagine (N) Amino acid 59 = Cysteine ( C) was replaced with arginine ( R) Amino acid 51 = Asparagine (N) was replaced with isoleucine (I) Amino acid 164 = Isoleucine (I) was replaced with leucine (L)

NCSI to NCNI to NRNI to IRNI to IRNL

5. How proteins are controlled/regulated:

Why do they need to be regulated?

Figure 4-35 Essential Cell Biology ( Garland Science 2010)

A. Multiple binding sites: cooperative binding is allosteric. The protein exists in 2 or more conformations depending on the binding of a molecule to the protein at a location apart from the catalytic site.

Figure 4-37 Essential Cell Biology ( Garland Science 2010)

B. Phosphorylation

ATP Substrate

Protein kinase has 2 ligand sites: One for ATP One for substrate protein

C. Nucleotide binding/hydrolysis

Figure 4-39 Essential Cell Biology ( Garland Science 2010)

Some proteins use GTP: bind GTP, hydrolyze to GDP + released Phosphate group

Some proteins use ATP: bind ATP, hydrolyze to ADP + released Phosphate group
Some proteins use CTP: bind CTP, hydrolyze to CDP + released Phosphate group

Figure 3-31 Essential Cell Biology ( Garland Science 2010)

Figure 4-42 Essential Cell Biology ( Garland Science 2010)

D. Proteolytic cleavage:

Low potency Botulinum neurotoxin

High potency Botulinum neurotoxin