BIO430: Protein Structure

Osteogenesis Imperfecta

"glass bones disease" -mutations in either alpha1 or alpha2 chains of type 1 collagen -characterized by fractures, skeletal deformities, dwarfism, perinatal death

Noncovalent forces important to protein conformation

*1. hydrophobic forces* -hydrophobic AA orient toward the inside in aq. environments -hydrophilic AAs orient out *2. Electrostatic forces, ionic bonds or salt bridges* -electrostatic bonds form between charged AAs *3. Hydrogen Bonds* -type of electrostatic interact. formed between a weakly acidic donor (-OH or -NH) and weakly basic acceptor (=O or -N-) *4. Van der Waal's forces* -transient, weak electrical attraction w/ important roles in protein-protein recognition -*antibody-antigen recognition, "lock and key"*

*Invariant Amino Acid* Position

*Gly in triple helix*

What sole AA has a side chain that can form covalent bonds yielding disulfide bridges?

*cysteine*

Types of Denaturation

-*heat:* increasing temp disrupts weak bonds and secondary structures -*pH:* changes ionization state of aa R groups, disrupting secondary structures -*chemicals:* detergents & chaotropic agents allow H2O to enter hydrophobic areas and disrupt normal folding

Secondary Structure

-*repeating structures that form segments in protein structure* -several types of secondary structure = *a-helix, b-pleated sheets, & reverse turn b-turn*

Secondary Structure: a-helix

-RIGHT handed helix -3.6 AA acids per turn -carbonyl oxygen *hydrogen bonded* to 4th amide H -AA R-groups orient OUT -center appears hollow- but actually full of atoms -*proline* is RARELY found in helices (*proline is an a-helix BREAKER*)

Prion Diseases

-bovine spongiform encephalopathies (Mad Cow's Disease) -scrapie (sheep) -transmissible spongiform encephalopathies (TSE-humans)

Protein Super Secondary Structures

-can consist of diff. types of elements @ diff. locations in the primary struc. -secondary struc. elements can bring distant portions of primary structure close together in space -*Motifs* = common super secondary struc. that do NOT predict the bio. func. (they are found in proteins/enzymes w/ dissimilar functions = ex. helix loop helix motif) -*Domains* = conserve, discrete parts of protein seq. that are folded units of secondary struc. and/or motifs (sometimes tertiary structures) - *PREDICT or IMPART a specific protein func.* and exist independently of the rest of the protein chain (ex. PDZ domain- anchors receptor proteins in the membrane to cytoskeletal components) -most proteins consist of several domains

Protein Denaturation

-denaturation is protein unfolding, usually with loss of biological activity -denaturation of proteins involves the disruption and loss of secondary/tertiary structures -denaturation reactions are not strong enough to break the peptide bonds, the primary structure (seq. of AA) remains intact

Review Q: Things that denature proteins?

-heat -pH -chemicals (chaotropes like Urea) -detergents like (SDS)

Review Q: Forces that stabilize protein structure?

-hydrophobic effect -hydrogen bonding -charge-charge interactions -disulfide bonds -metal ions

Secondary Structure: Loops and Turns

-loops and turns redirect the polypeptide backbone -a *turn* is a "tight" change in backbone direction -a *loop* can be more meandering -*GLYCINE and PROLINE* are very common AA in loops and turns

Fibrous Proteins: Collagen

-major structural protein; skin, bones -different types of collage in different tissues -part of extracellular matrix outside of cells -gelatin is denatured collagen (jello)

Protein Quaternary Structure

-most functional protein consist of multiple polypeptides -polypeptides may be identical or different -usually polypeptides interact in symmetrical manner -pic is an example of *dimer protein* (Cro protein from bacteriophage gamma)

Tertiary Structure

-overall folded conformation of polypeptide *many physical forces are important to protein tertiary structure:* -*hydrophobic forces*: hydrophobic resudes orient to inside, hydrophillic orient out -*electrostatic forces*: ionic bonds, salt bridges -*Van Der Waals Radii* -*H-bonds* -*Disulfide Bridges* ex. CD4 protein (4 similar yet distinct domains in one polypeptide)

Protein Domains

-part of polypeptide chain that *can independently fold* into a tertiary struc. -domains often are units of func. -proteins may contain 1+ domains

Secondary Structure: B-pleated sheet

-polypeptide chains side by side -B-strand is a single pass of polypeptide in a sheet B-strands can be: *1. antiparallel* -atoms involved in H-bond directly opposite each other -usu. has a hydrophobic & hydrophilic side *2. parallel* -atoms involved in H-bond are slightly skewered -tend to have hydrophobic residues on both sides of sheet carbonyl oxygen *hydrogen bonded* to amide hydrogen

Four Levels of Protein Structure

-primary structure: seq. of AA by peptide bonds -secondary structure: folded 2D struc. of primary -tertiary structure: 3D shape of polypeptide -quaternary structure: 3D shape of multi. interacting polypeptides *primary structure determines all other levels of protein structure*

Defects in Collagen Synthesis

-scruvy -osteogenesis imperfecta -ehlers-danlos syndrome

Myoglobin

-single polypeptide (153AAs) containing several a-helicies -blue helix is tucked into a crevice between red helices and the protein is compact -*heme* group fits into a pocket between the red and blue helices

Supersecondary Structures: Motifs

-smallest protein conformation unit (may be func.) -globular proteins constructed by combining second structural elements examples are: - aa (helix-loop-helix), BaB, B-hairpin - B-barrels (B-strands form a barrel) -Rossman fold (nucleotide binding site) -*leucine zipper* (mediates transcription factor dimerization) -*zinc finger (DNA binding motif)*

Quaternary Structure

-subunit structure -*interaction between 2+ subunits* -can be hetero- or homo-polymers -*same physical forces drive tertiary and quaternary struc. formation* -subunits may be linked by interchain disulfide bridges (more so in fibrous/structural proteins, rarely in globular proteins)

Scurvy

-vitamin C deficiency -*prolyl and lysyl hydroxylase* enzymes; require vitamin C as a coenzyme

Conservative vs Nonconservative AA substitution

1. Point Mutations: Change in a single base pair -silent: no change in the encoded AA -missense: change from one AA to a different AA >> *Conservative:* Glu (GAG) to ASP (GAC) >> *Nonconservative:* Glu (GAA) to Val (GUA) -nonsense: addition of premature stop codon

Some a-helices have a ____ nature

Amphipathic -shown in a helical wheel rep. of a-helix of alcohol dehydrogenase.

_____ forces are important to protein conformation

Covalent -arise when two atoms share E- - ex. peptide bonds and disulfide bridges

What is necessarily characterized by a covalent linkage?

Disulfide Bond

Which of the following AA has a net negative charge at a physiological pH (~7.4)

Glutamate

Protein Quaternary Structure ex. Hemoglobin

Hb is a a2B2 *tetramer* with two sets of monomers of alpha (magenta) and 2 monomers of beta (yellow) polypeptides arrange symmetrically -*alpha and beta monomers* are the product of different genes.

All of the following would disrupt quaternary structure except..

High temperatures and adding BM urea would disrupt the structure except for treating with ascorbic acid (VitaminC)

What maintains the secondary structure of a protein?

Hydrogen Bonds

Protein Conformation (shape) is crucial to _____

Protein Function

Tropocollagen

basic structural subunit of collagen -structure is 3 intertwined helices; *triple helix* -left-handed helix about 1000AA (*not an alpha helix*) -helix contains 338 Gly-Xaa-Yaa repeats (X: often Pro, Y: often HyPro/HyLys) -elongated approx. 3AA/turn -*glycine required for triple helix to form*

Review Q: Protein Reduction?

breaks disulfide bonds between cysteine -ex. DTT and B-mercaptoethanol

Protein Reduction

different than denaturation -*reducing agents* (B-mercaptoethanol or DTT) break distilled bonds between cysteines -when ribonuclease was treated with chaotrope (urea) and a reducing agent (B-merca..) it lost its enzymatic activity -when the denatured ribonuclease was dialyzed to removed the urea and the B-me the protein renatured, restoring its activity -thus denatured, linear proteins only function when folded correctly

Ehlers-Danlos Syndrome

family of disease encompassing a variety of collagen processing defects -ex. stretching of skin or bending fingers

What does NOT influence protein shape?

peptide bonds -because it makes primary sequence but nothing about building

Protein Folding and Stability

primary structure determines the final tertiary and quaternary structure *forces stabilizing protein structure* -hydrophobic effect -hydrogen bonding -charge-charge interactions -disulfide bonds -metal ions

Chaperones and Protein Folding

proteins can self assemble but in vivo folding is often facilitated by proteins *chaperones* are binding proteins which assist folding -chaperones cause misfolded/unfolded proteins to remain unfolded rather than aggregate -more then one type of chaperone may at simultaneously and sequentially in the folding of a single protein -chaperones are specific for specific protein synthesis pathways (cytosolic vs. mito vs. endoplasmic reticulum)

Sickle Cell Anemia

result of a point mutation (autosomal recessive - heterozygote is asymptomatic) -causes a *Glu>Val* replacement at position 6 of the B-chain (switches charged AA to a hydrophobic AA) -decreased solubility upon deoxygenation >> causes polymerization of Hb S >> forms long polymers >> causes RBCs to deform (sickle) >> nickel cells rupture in tight places in circulation -mutations can be detected by electrophoresis >> mutation causes a change in Hb charge