M114 part 1

Phi and Psi backbone

stabilized by backbone hydrogen bonds (180 degrees beta), Phi (between C-N), Psi(between C-C), rigid and can only rotate

Cis and trans peptide bonds

They got to stay away in trans structure, in cis structure it has double bond(proline) becomes 2-D makes a u-turn

Types of secondary structure

Repetitive: intera peptide H bond helics(3_10—>a—>Pi), Interpeptide H bond helices structure: B strands, antiparallel or parallel B sheets

Non Repetitive: random, happen to be there

Beta Turn

2 hydrogen bonds, stabilize it, minimum 4 amino acids, secondary structure

Helics with intra peptide H bonds (folds H bond within)

3_10(1AA+AA4) distorted, alpha(AA1+AA4) NEAR PERFECT, Pi(AA1+AA5)distorted

Alpha Helix

1. very stable

2. number of H bonds-total aa-4

3. 180 degrees for every H-bond

4. favorable delta S

5. positive at NH termibak end

6. negative at CO terminal end

1. 5.4 A per turn, 3.6 residues per turn

2. upside down Xmas tree

3. NPPN

Beta sheets

1. Extended, pleated structure

2. H-bonds between adjacent β-strands

3. Parallel = same N→C direction

4. Antiparallel = opposite N→C directions

5. Antiparallel H-bonds more linear/stronger

6. Side chains alternate above/below sheet

7. ~3.5 Å rise per residue

8. Prefer V, I, C, Y, W, F, T

9. β-sheets usually right-handed twist

10. Parallel = angled H-bonds; antiparallel = straighter H-bonds

Ramachandran plot

Protein Structures

Barrel vs twist

Similar secondary structure elements, comp etely different tertiary structures

Tertiary structure

Tertiary Structure: Stablized by Interior Packing

Stablizing the Protein Fold

Add: reversable :disulfide bridge cofactor binding glycosylation phosphorylation acylation ADP-ribosylation carbamylation N-acetylation

Stablization Strategy

By disulfide bond bovine pancreatic trypsin inhibitor or By metal coordinate covalent bonds Bacterial subtilisin

Stablization Strategies

Cofactor Binding: NADP or NADPH bind to active site and change tertiary structure

Intermediate Levels of Protein Structure

Primary • Secondary • Domain — A compact assembly of secondary structures that fold into a stable entity. • Tertiary: it can consist of multiple domains • Quaternary

Motif

Definition: — Characteristics in structure or sequence, may be a whole domain or an arrangement of secondary structure element

A Motif can be as little as three amino acids (The Catalytic Triad of protease, D-H-S)

motif can consist of a few secondary structures

Helix turn helix, DNA binding motif, Major groove

Homeo-domain:

helix- turn-helix motif that binds DNA and determines our development

2 major alpha domain motifs

1. cytochrome(A.S top and bottom)

2. Globin fold( A.S inside near top)

The Hairpin Motif

No active site

Up and Down motif

A.S at top or bottom and 2 domains

Plain b Barrel

active site in the middle

The Greek Key Motif

top or bottom A.S

The Jelly Roll

A.S top or bottom

Anti-parallel a/b Domain Motif The Saddle

A.S under beta sheet

Anti-Parallel a/b Domain Motif

Metal Ions and Disulfide Cross Links

The Zinc Finger Motif

a helix + antiparallel b 2 His + 2 Cys to one Zinc Binds DNA major groove

a+b Domains: Parallel b Strands Connected by a helices or loops

The Parallel Beta Sheet

TIM barrel or twist

The TIM Barrel

only 8 strands to have sufficient twist so that the end strand can roll back on itself and close.

The b Twist

The alternating a / b sheet, usually < 7 b Common for nucleotide binding proteins (NADP, ATP or GTP)

Quaternary Structure

Quatemary Structure Definition — Quaternary structure refers to the number, type, and arrangement of subunits, which self-assemble into an oligomer in solution under physiological conditions. • Subunit Definition — Subunits are non-covalently associated polypeptide chains that are each folded into a structure that is a stable entity by itself. Subunits are independent units with respect to structure but not function.

Surface complementation is key to quaternary structure formation

One single mutation chan ed the quaternary structure of

In beta subunits change from ASN to VAL or Lue to Val (hydrophobic)

Motion Found in Proteins

Fluctuations, collective motions, triggered conformational changes

Five ways to represent a protein structure

wire, ribbon, ball and stick, space filling, surface

Mutiple confirmations

Purified protien—> NMR spec—> resonance assignment—> protien structure

Four major steps in crystallization

Obtain large amounts of pure protein samples • Choose a protein buffer in which the protein is both soluble and stable • Bring protein solution to supersaturation where spontaneous nucleation can take place • Crystal growth now begins

Sitting drop vapor diffusion

slowly evaporating away the water so protien concentration goes up and salt concentration goes up

How to Judge a Crystal Structure

Resolution (better than 3 A) • Rmerge of data (< 10% overall, <50% highest resolution shell) • Rfree of model (<25%) • Ramanchadran plot (no disallowed region { SUM UP TO 100%}

Membrane Proteins: all-alpha or all-beta

Trans-membrane proteins

All-beta (polyto ic): porin, transporter • All-alpha: — Rhodopsin — G-protein coupled receptor (GPCR) — Channel — Transporter/pump, e.g. ATP Binding Cassette (ABC) — Photosynthesis system — Protease, ATPase, oxidoreductase, respiratory enzymes — Toxins

Matching the membrane thickness with a helix

1 residue rise in helix: 1.5 To span the membrane, we need 30/1.5 = 20 aa

Matching the membrane thickness with ß strand

It has to be anti-parallel Inter-residue distance in ß stra porin : 3.5 A span the memb ne, 30/3.5 = 8 aa

The Calcium ATP se, El state

4 domains: 1st transmembrane(connects Ca²+ charged ions), 2nd A domain, 3rd P(phosphate) domain, 4th N domain (ATP bond)

The Calcium ATPase E2 state

ATP bind E1—>E2 thru phosphorylation, close to active sit (p) change of confirmation hairpin(N) and at bottom of N is ATP bind

Pumping Ca

The selectivity filter

How did K+ Channel prefer K+, not Na+

K+-protein interaction > desolvation penalty —> Go through channel

Na+-protein interaction < desolvation penalty —> Blocked

Transported by Repulsion

Selection filter can bind four K+ Charge-charge repulsion: push K+ outwar

What is Drug Design?

A multi-step process that combines many scientific disciplines to create chemical compounds that can benefit our health

Structure-Based Drug Design:

Docking & Scoring.: 1) A protein structure, 2) A small molecule database, 3) a reliable software

Criteria for Druggable Pockets

1. shape—> cave or minimal depression

2. hydrophobic interior—> higher than avg of surface exposed hydrophobic area

3. Size= usually 200 to 600 A

Fill the cavity w/ binding

Once hit 500 things dont work

binding specificity: alcohol dehydrogenase (blue, not used to increase amount of alcohol pieces)—> isozyme: red high alcohol processing—> isozyme binds to active site (5 H-bond 10^5 between drug and enzyme)

blue: isophone 2= lost 2H-bonds [3H bonds 10^4 pocket= drug doesnt work)

Fragment based method

1. compound almost not exactly like drug lead

2. separate drug—> chemiotaxis—>into 3= fit in enzume binding pocket

3. Benzyne—> delta G—> carbon group=then choose what docks in cavity[chemiotaxis]

[FlexX—> join in LUDI—> DOCK]

HIV protease:HIV Protease: Dimeric, Acid Protease

1. RNA virus= HIV protease—> breaksdown peptide bond of protein

2. protease inhibitor—> unbound—> less energy to adapt to binding site—> H-binding—>active site—>H-bond w/backbone—>improved binding—>mutation= weaken binding

3. Homodimer—>top(beta hairpin—>confirmational change=open for drug to enter/bind)

4. Asp(binding site), flap (conf.change), red color acid over Nose (active site)

5. tetrahedral

Two elements of a good drug

1. enzyme recruits own substrait= protein (polypeptide) memic

2. transition state analog (TSA)

Specificity pocket

every protease has a favored amino acid [Trypsin—> cuts after positivily charged residues, chymotrypsin—> cuts after larg hydrophobic residues, Elastas—> cuts after small hydrophobic residues] (lys, arg, asp, benzene ring)

Break scissile bond

between p1 and p1’= create procket to accomedate

good drug—> transition state (look alike tetrahedral)

enzyme binds tightest to transition state (high)=lower than 100,000 fold w/ enzyme

The drug needs to be tetrahedral

HIV protease #1 factor

pocket for Phe group —>and Pro group—> drug need Pro and Phe(p1 and p1’)

Combine drugs

cocktail to confuse HIV protease

COVID Protease

3 CL6^pro, M^pro

homo dimer—> 2 active sites

SARS- COV2: P1—>Q, P1’—> S

N3 p1 and p1’ (glu, sre)—> cavity phe ring

well defined tightly bond to active site of enzyme

Substrate pockets

fully different only N3 and SARS-COV2 can bind togther

virtual screening: IC—> invitiro assay

1:1 suicide inhibitor

drug vs protien

kill 1 of each other

Mpro Substrate Specificity

1. cut between Q and S

2. Q= Glutamine, S(serine), p2 = Leucine

3. P1’= T, S or A

What is a suicide inhibitor?

An inhibitor that forms covalent bond(s) with the active site Cys or Ser. In this case, a Michael Addition • First one is penicillin

. Explain, in one sentence, how sodium channel would bind only sodium but not potassium

Na solvent penalty < Na-channel interaction, while K-solvent penalty > K-sodium channel interaction

Backbone Hydrogen Bonds

α-helix: H-bonds = n − 4

50 aa → 50 − 4 = 46 H-bonds

Antiparallel β-sheet: 8 strands × 8 aa → 49 H-bonds

Secondary Structure Prediction

α-helix: E, Q, A, L, M, K, R

β-strand: V, I, C, Y, W, F, T

Loop/turn: G, P, N, S, D

VSVSVSVS → antiparallel β (professor's key)

GPGGP → loop (Gly/Pro favor turns)

Hydrophobic Effect

Nonpolar exposed to H₂O → ordered water → ΔSsolvent ↓ → ΔG ↑ (positive) → exposure unfavorable.

ΔG → Binding Constant

ΔG = −RT ln K

R = 0.001987 kcal/mol·K; T = 298 K

Fold change = e(ΔΔG/RT)

Loss of 10 H-bonds (+14 kcal/mol) → K decreases ≈ 10¹⁰-fold

cDNA → Protein Molecular Weight

MW (kDa) = [(bp ÷ 3) × 110] ÷ 1000

40 kb → 40,000 ÷ 3 × 110 ÷ 1000 ≈ 1,467 kDa

Shortcut: kb × 36.7 ≈ kDa

β-sheets vs. α-helices

• α-helix H-bonds form within the same continuous helix (residue \(i\) to \(i+4\)).

• β-sheet H-bonds form between neighboring strands, which can be far apart in the primary sequence.

Parallel vs. Antiparallel β-Sheets

Feature	Parallel	Antiparallel
Direction	Same → →	Opposite → ←
H-bonds	Angled	More linear
Stability of H-bonds	Generally less optimal geometry	Generally more favorable geometry
Connections	Often longer crossover loops	Can form short β-hairpins

The Four Strategies of Signal Transduction:

Specificity, Amplification, Adaption, Integration

Six Mechanisms of Signal Transduction

GPCR, receptor tyrosine kinase, guanylyl cyclase, adhesion receptor, gated ion channel, nuclear receptor (no membrane bound)

Kinase inhibitor as cancer drug design:

1. ATP mimic

2. Lock kinase in an inactive conformation

SH3 Domain: binding Pro-rich helix

no cavity, SH3 bind proline, antiparallel beta sheets

SH2 Domain: binding P-Tyr

SH2—> PHOSPHORALATED TYROSIN

An protein. Mixed: electrostatic + H-bonds + hydrophobic

PTB

Binds to P-Tyrosin, ß-barrel with a helical cap Mixed: electrostatic + H-bonds + hydrophobic

PH: binding IP3 (membrane)

binding IP3 (membrane)

Two layers Of anti-parallel II-sheets with a helical cap.

Diabetes

Why did Mario Puzo's eyesight decayed? diabetes affecting eyesight

• What may aggravate de res ion of Hemingway? toxic iron deposit and hemochromtosis

• Think of the above in terms of our body organs that mainly use glycolysis (aka the breakdown of glucose) for energy? eyes, brains and kidneys

INSULIN

Insulin receptor gets Tyr phosphorylated IRS Grab2, SOS Then comes Ras the G protein Raf MeK, Erk, the MAP kinase

The auto-phosphorylation Of insulin receptor

Insulin binds to alpha subunit

beta receptrs are autophosporalted to kinase

and active site protected by facing the interior of protein (hydrophobic) with 3 tyrosines (-6 charges)

IRS

Has PTB domain and binds P-Tyr

Grb2 is the adaptor

1.SH2 binds to IRS1, SH3 binds to SOS

SOS is RAS GTP exchange factor(sos activates ras basically)

3. pro-rich to bind SH3 in Grb2 —> switch1: activated by cdc25 and switch2 activiated by REM domain

4. both switch1/2 need to face in = interacts with Raf 1[ if GTP becomes GDP then switch out and no raf1 interaction]aaaaaaaaaaaaaaaaaaaaaa

aadrugs targeting at the Ras protien?

Cancer, SADDR (sotorasib, adagrasib, divarasib, daraxonrasib, rmc-6236

Ras-GTP: allows Ser-259 P-free

RAF activated by GTP—> MEK—> ERK

INFLUENCE OF INSULIN

ACTIVATED: active transport, glycolysis, glycogen synthesis, lipid synthesis, protein synthesis

INHIBITED: gluconeogenesis, lipid breakdown

Why can’t DNA hydrolyzed by RNase.

Bec it doesn’t have 2 2’OH which is then impossinle to form ring and hydrolysis rxn

Why are vanadates good RNase inhibitor

enzyme inhibotor, trnasition metal and rnase inhibitor

RNA BREAKS EASILY BY HIS AND FIVE MEMBER RING

Self-splicing RNA. NO amino acids for acid-base catalysis so how does it work?

1. domain chape(loop to alpha helix)—> 1 phosphadiaster bond broken and rna catalyzes the rxn

The Proposed Transition:

Ribozyme, ribonucleoprotein enzyme, protein enzyme with nucleotide coenzyme

Metal Ion Catalysis

General acid base= HO-H + Mg, Lewis acids(O+Mg), Electrophilic catalysis (P+ Mg)

Ribozyme Pharmaceuticals, INC

Angiozyme, Hepatozyme, Herzyme

LUPTAKE LAB

he used TSA (best binder to an enzyme)

Ribosome race results

Tom Steitz won by 2.4 A but Ada Yonath first to crystalize

The ribosome has 4 binding sites

E, P, A and mRNA not found it is i the small ribosomal subunit

1. aa+ATP+tRNA—>aa-tRNA+AMP

ATP activate, and adenylated AA to make ribose to adenine