Protein Structure & Function!

Protein Structure ! !! ! ! !Learning Objectives! ! ! Primary structure! ! Protein folding! ! Elements of secondary structure (! helix / " sheet)! ! Tertiary structure (domains & modules)! ! Multi-subunit protein complexes! ! Self assembly of protein complexes!
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Proteins are linear polymers of !-amino acids joined by peptide bonds. ! Peptide bonds are formed when the ! carbonyl group of one amino acid condenses with the ! amino group of another to to form a substituted amide. #G0 = 2.2 kcal/mol

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Proteins are polypeptides!

04_02_polypeptide back.jpg!

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ligand binding & catalysis phosphoryation methylation, acetylation, ubiquination, sumoylation, ! glycosylation conformational stability

Primary Structure! A source of tremendous structural diversity"

20 Common Amino Acids (AA) ! 20n possible peptides of n AA Example 1 8 10 AA peptides are common signaling molecules There are 208 = 25,600,000,000 possible unique 8 AA peptides Example 2 Average protein ~ 300 AA ! 20300 = 10390 > > # atoms in universe !

However, not all amino acids are equally represented in proteins C + W + M = 5% L + S + K + E = 32%

Somehow, a protein manages to fold into a single stable conformation!

Protein conformation is stabilized by non-covalent bonds!

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Table 2-1 Essential Cell Biology ( Garland Science 2010)

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The Hydrophobic Effect

Entropically driven exclusion of non-polar solutes from aqueous media "!Thermodynamically Unfavored

"!Thermodynamically Favored

* ! Many proteins have a hydrophobic core!

Reversible Denaturation!

Figure 3-3a Molecular Biology of the Cell ( Garland Science 2008)

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Reversible Denaturation!

All of the information required to direct proper protein folding is contained in the primary structure
Figure 3-6a Molecular Biology of the Cell ( Garland Science 2008)

Information required for proper folding of proteins " is encoded in the AA sequence!

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Proper folding is thermodynamically driven, resulting in the conformation of lowest energy. Folding is a step-wise process that gives rise to distinct intermediate structures (e.g., molten globule).

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Proper folding of proteins brings into position non-adjacent amino acids that participate in protein function! ! Enzyme active sites! Receptor binding sites! Protein-protein interaction surfaces!

active conformation!

Figure 3-37a Molecular Biology of the Cell ( Garland Science 2008)

4o

Multimeric Complex Protein Domains

3o

(functional / structural)

2o 1o

Secondary Structural Elements

(! helix / " sheet)

Amino Acid Sequence

Graphical representations of Ras (a GTP-binding protein)!

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C! trace (wireframe)

ball & stick

+ -

schematic (ribbon)

(water accessible) surface

Elements of Secondary Structure!

$ !

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random coil helix

loops

$ "

sheet

disulfide bridge turns

Ribbon model of the binding domain of a major histocompatibility complex (MHC) protein

Secondary Structure! Local conformational stability"

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Together the ! helix and " sheet account for 60 percent of the secondary structure of polypeptides. These structural elements are stabilized through hydrogen bonding.

! Helix

" Sheet

Secondary Structure" The ! Helix!

3.6 residues per turn AA side chains directed outward Stabilized by hydrogen bonds *between amide NH & carbonyl CO* Can have amphipathic character

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cartilage oligomeric matrix protein (COMP)

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04_15_ahelix_lip_bilayer.jpg!
18 22 AA are sufficient to traverse the plasma membrane

Secondary Structure" The " Pleated Sheet!

Composed of " strands arranged in either parallel or anti-parallel orientation Strands are stabilized by hydrogen bonds AA side chains project toward alternate faces of the sheet Can have amphipathic character

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" Sheets come in two conformations both are common

Anti-parallel Sheet

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Parallel Sheet

Linus Pauling (1901 - 1994)" Father of Structural Biochemistry!

Proposed the structures of both the ! helix and " pleated sheet

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Only person to receive both the Nobel Prize (Chemistry; 1954) and the Nobel Peace Prize (1963. Linus Pauling (~1958) Lecturing at CalTech www.paulingexhibit.org Was the first to propose a helical structure for DNA, but got the details wrong

Coiled-Coil !

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04_16_coiled-coil.jpg!
8 4 1

heptad

4 1

Coiled-Coil!

Associations of two ! helices bound together by hydrophobic interactions between non-polar AA present along one side of each helix. Stabilizes protein-protein interactions Commonly found in dimerization domains of DNA binding proteins such as c-Jun. Conserved heptad (1-4-8) sequence LASTANM LREQVAQ L Leucine Zipper

Domains are stable regions of a protein consisting of groups of secondary structural elements

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As nascent polypeptide emerges from the ribosome 1.! Secondary structures form 2.! Domains fold

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Folding is completed after release of protein from ribosome

Domains are stable regions of a protein consisting of groups of secondary structural elements

04_20_protein domains.jpg!

cytochrome b562

NAD binding domain immunoglobulin of lactic dehydrogenase variable domain

Green Fluorescent Protein: a " Barrel

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Common Protein Modules

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Modules are domains that appear in many different proteins. Protein evolution appears to involve recombination of modules to yield proteins with new functionality

Fibronectin (fragment)!

Fibronectin type 3 modules !

Figure 3-17 Molecular Biology of the Cell ( Garland Science 2008)

Modular Mix-N-Match gives rise to proteins with new combinations of properties

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EGF domain Trypsin domain (serine protease) Kringle domain GLA domain

Figure 3-15 Molecular Biology of the Cell ( Garland Science 2008)

Tertiary structure is preserved within protein families!

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Conserved Regions

Immunoglobulin Molecule!

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Variable domain is comprised of both chains

Hemoglobin

heterotetrameric complex (!2"2)

Neuraminidase forms a homotetrameric protein complex

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Blue and orange domains bind: determines geometry

Globular proteins can assemble into complex structures

Relationship between binding domains sets shape

Actin filaments form spontaneously in the presence of globular actin subunits and ATP

Figure 3-25 Molecular Biology of the Cell ( Garland Science 2008)

Globular protein subunits bind to form complex structures

(e.g., tobacco mosaic virus)

Figure 3-29 Molecular Biology of the Cell ( Garland Science 2008)

Tobacco Mosaic Virus

Tobacco Mosaic Virus 158 AA

2130 subunits

Viral Capsids

Spontaneous Self-Assembly

Protein Function! ! ! !Leaning Objectives! ! ! Non-covalent modication of protein conformation (Allosterism)! ! Covalent modication of protein conformation! ! Molecular switches & signal integration! ! Molecular chaperones! ! Misfolded proteins (very bad!)! ! Targeted (ubiquitin-mediated) proteolysis!

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Ligand binding site accepts, and interacts with another molecule through formation of multiple non-covalent bonds

Induced Fit
Figure 4-27 Essential Cell Biology ( Garland Science 2010)

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Proper folding of proteins brings into position non-adjacent amino acids that participate in protein function! ! Enzyme active sites! Receptor binding sites! Protein-protein interaction surfaces!

active conformation!

Figure 3-37a Molecular Biology of the Cell ( Garland Science 2008)

Typical cyclic AMP binding site

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

Lysozyme is a polysaccharide hydrolase that attacks the cell wall of certain bacteria, acting as an antibiotic

Figure 4-30b Essential Cell Biology ( Garland Science 2010)

Proposed mechanism of Lysozyme-catalyzed reaction

Coordinated Acid / Base Catalysis

Proposed mechanism of Lysozyme-catalyzed reaction

Figure 4-31 Essential Cell Biology ( Garland Science 2010)

Proposed mechanism of Lysozyme-catalyzed reaction

Figure 4-31 Essential Cell Biology ( Garland Science 2010)

Enzyme (E) binds substrate (S) to form complex (ES), catalyzes hydrolysis (EP) to form severed products (P)

Catalysts increase the rate of product formation, AND are unchanged by the reaction

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Michaelis Menten Kinetics

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Vmax ! catalytic power KM ! substrate affinity

Figure 3-26b Essential Cell Biology ( Garland Science 2010)

Michaelis Menten Kinetics

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v=
When [S] = KM

Vmax 2

Figure 3-26b Essential Cell Biology ( Garland Science 2010)

Michaelis Menten Kinetics

v=
When [S] = KM

Vmax 2

Figure 3-24 Essential Cell Biology ( Garland Science 2010)

Lineweaver Burk Plot

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Figure 3-26c Essential Cell Biology ( Garland Science 2010)

Know your Enzymes

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

Prosthetic groups extend the capabilities of enzymes

heme

11 cis- retinal

Allosteric Regulation of Protein Conformation Hemoglobin

Cooperative Binding of O2 percent saturation

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pO2 (mm Hg)

deoxyhemoglobin

oxyhemoglobin

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T State

R State

Allostery involves the binding of a modulator to a site that is distinct (and often at a distance) from the active site

Positive Allosteric Regulation

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

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Allosteric regulation of a metabolic pathway Negative Allosteric Regulation

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Figure 4-34 Essential Cell Biology ( Garland Science 2010)

aspartate carbamoyltransferase: an allosteric enzyme complex controlling pyrimidine biosynthesis !

CTP!

12 subunits! C6R6!

aspartate carbamoyltransferase!

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Figure 4-36 Essential Cell Biology ( Garland Science 2010)

Controlling protein function by reversible phosphorylation Covalent Modification

Figure 4-38a Essential Cell Biology ( Garland Science 2010)

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Controlling protein function by reversible phosphorylation Phosphorylation state of a phosphoprotein depends on relative activities of kinase and phosphatase. Many phosphatases are constitutively expressed default is dephosphorylation

Figure 4-38a Essential Cell Biology ( Garland Science 2010)

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Proteins can be activated or inactivated by phosphorylation

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Figure 4-38b Essential Cell Biology ( Garland Science 2010)

GTP activates small GTPase signal proteins G-proteins

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Some monomeric G-proteins require regulatory proteins -- GTPase Activating Protein (GAP) -- Guanine nucleotide Exchange Factor (GEF)

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GTPase Activating Protein

Guanine Nucleotide Exchange Factor

Elongation Factor Tu (EF-Tu) - a GTPase switch protein

Elongation Factor Tu (EF-Tu) - a GTPase switch protein

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Prokaryotic protein that escorts amino acyl tRNA to the A site of the ribosome. If codon-anticodon binding occurs, GTP is hydrolyzed, EF-Tu releases the t-RNA

Some proteins integrate signals through subunit interactions

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!"#$%&%'()$*+,-#()$.#-#+/()#0$-"#$0120-+3-#$0*#%(4%(-&$,5$-"#$ 6()30#7$3).$(0$+#81(+#.$5,+$6()30#$3%9:39,) $

D#:#+3'$.(I#+#)-$J&%'()0$3+#$*+,.1%#.$.1+()<$-"#$%#''$%&%'#$

;(<1+#$=>?=@$!"#$%&'$()!*+#$#,-!#.!/0%!1%$$$AB$C3+'3).$D%(#)%#$EFFGH$

Some proteins integrate signals through subunit interactions

Cyclin-dependent Protein kinase

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Peyton Rous (1911) Cell-free extract of chick sarcoma causes tumor formation = virus Rous Sarcoma Virus RSV carries the src oncogene src (tyrosine) kinase src encodes a mutant tyrosine kinase

Src Homology Domains

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SH3 domains bind to prolinerich sequences

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SH2 domains bind to phosphotyrosine residues

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Figure 3-69 Molecular Biology of the Cell ( Garland Science 2008)

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SH2-pTyr

SH3-Pro-rich

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1. Dephosphorylation of tyrosine residue on SH2

1.! Dephosphorylation of tyrosine residue on SH2 2.! Binding of Activating Ligand (protein) 3.! Phosphorylation of the Catalytic Loop

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Figure 3-69 Molecular Biology of the Cell ( Garland Science 2008)

2. Binding of Activating Ligand (protein)

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SH3-Pro-rich SH2-pTyr

Figure 3-69 Molecular Biology of the Cell ( Garland Science 2008)

3. Phosphorylation of the Catalytic Loop

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Some proteins integrate signals through subunit interactions

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