BS400 Molecular Biophysics BT808 Principles of Biomolecular - - PowerPoint PPT Presentation

• BS400 Molecular Biophysics

BT808 Principles of Biomolecular Interaction and Recognition

Course material will be made available on moodle moodle.iitb.ac.in

• To learn about the factors that are responsible for

the conformation and assembly of biomolecules and recognition and interactions of biomolecules

Objective

EMPHASIS Manifestation in biological systems Current status and limitations Recent and classic examples

• This course vis-à-vis others in the curriculum

An imaginary hybrid of telescope and microscope We see things differently when we change the depth of focus

• Prof. Sudesh Balan, IDC, IIT Bombay: Depth of Focus video

vis-a-vis (meaning from an online dictionary): (1) in relation to (2) as compared to

• Outline
• 1. Sequence - {Folding} - Structure - Dynamics - Function paradigm:

perspective for the course

• 2. Molecular structure: representation, conformational changes,

conformational ensembles, conformer selection

• 3. Steric effect or hard-sphere approximation: preferred (allowed)

versus not-preferred (disallowed) conformations; application to monosaccharides and peptides; Ramachandran map ... continued

paradigm (meaning from online dictionaries): (1) The generally accepted perspective of a particular discipline at a given time (2) A framework within which theories, laws, and generalizations and the experiments performed in support of them are formulated

• Outline
• 4. Non-covalent interactions: types, relative strengths, examples

(from biological systems) of their manifestation and consequences

• 5. Entropy: standard entropy of approximation, entropic and

enthalpic cooperativities, enthalpy-entropy compensation, hydrophobic effect, conformationally constrained molecules and protein design

• 6. Stability and folding of proteins: measuring conformational

stability, theories of protein folding, φ φ φ φ value analysis

• 7. Lipids: phase transitions
• 8. Binding: Specificity and affinity

• Reading material

Books Recent editions of general Biochemistry books include ■ a chapter on non-covalent interactions in Biology ■ structural aspects of biomolecules Important Spend at least 30-45 minutes after EVERY class

• Go through slides / any points you might have noted down,
• Form small groups and discuss
• Make an attempt to write down in a few “bullet points” the

concepts discussed or key points conveyed or “take home message” of that class

• Clarify doubts either in the next class or in person ASAP

• “Extra” reading material
• 1. Books
• Proteins. Structure and molecular properties by Creighton

Structure in proteins by Kyte

• 2. Original research papers and review articles

Will give you the reference when we discuss the paper

• Holidays and “adjustments” this semester...
• 1. Friday January 6 - at scheduled time

(KReSIT Seminar Hall, 3rd floor)

• 2. Tuesday March 20 - Friday time-table
• 1. Friday February 10
• 2. Friday March 23
• 3. Wednesday April 4 - Thursday time-table
• 4. Friday April 6

• Evaluation for BS400

Quiz 1 15 marks 26th or 28th Jan. Mid-sem. 30 marks February 20-25, 2012 Quiz 2 15 marks 23rd or 24th March End-sem. 40 marks April 20-May 2, 2012

• Evaluation for BT808

Quiz 1 10 marks 26th or 28th Jan. Mid-sem. 30 marks February 20-25, 2012 Quiz 2 10 marks 23rd or 24th March Paper presentation 10 marks 2nd week of April End-sem. 40 marks April 20-May 2, 2012

• Outline
• 1. Sequence - {Folding} - Structure - Dynamics - Function paradigm:

perspective for the course

• 2. Molecular structure: representation, conformational changes,

conformational ensembles, conformer selection

• 3. Steric effect or hard-sphere approximation: preferred (allowed)

versus not-preferred (disallowed) conformations; application to monosaccharides and peptides; Ramachandran map ... continued

• 2009

Ribosome V Ramakrishnan, T A Steitz, A E Yonath 2003 Channels P Agre, R Mackinnon 2002 NMR methods K Wuethrich (0.5) 1991 NMR methods R R Ernst 1988 Photosynthetic RC J Deisenhofer, R Huber, H Michel 1985 X-ray direct methods H A Hauptman, J Karle 1982 X-ray electron microscopy A Klug 1974 Statistical mechanics P J Flory 1969 Concept of conformation D H R Barton, O Hassel 1966 Molecular orbital methods R S Mulliken 1964 X-ray, 3D structures D C Hodgkin 1962 X-ray, 3D structures M F Perutz, J C Kendrew 1958 Structure of insulin F Sanger 1962 Structure of DNA F H C Crick, J D Watson, M H F Wilkins

Importance of structure in Biology

• Sequence - Folding - Structure

GSAMARTEKIYIYGASGHGLVCEDVAKNMGYKECIFLDDFKGMKFESTLPKYDFFI AIGNNEIRKKIYQKISENGFKIVNLIHKSALISPSAIVEENAGILIMPYVVINAKA KIEKGVILNTSSVIEHECVIGEFSHVSVGAKCAGNVKIGKNCFLGINSCVLPNLSL ADDSILGGGATLVKNQDEKGVFVGVPAKRM Campylobacter jejuni PglD (an acetyltransferase) different views of the same structure PDB id 3BSY (chain A)

• Sequence - {Folding} - Structure

MSEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDFVTE TPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTAEEDHVD LSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSKRKP Cyclin dependent kinase inhibitor p21 (SwissProt accession no. P38936.3)

“folds” after it interacts with and binds to its target May fold differently on binding to different targets 3D structure ? Intrinsically disordered protein Natively unstructured protein

• J. Mol. Graphics Model. (2001) 19:26

• http://conservativetshirts.info/wp-

content/uploads/2011/09/harry-potter- giveaway-LA-11-15-101.jpg http://mjfredrick.files.wordpress.com/2011/03/ harry-potter-with-wand-wallpaper.jpg

Static versus dynamic

Still picture (a photograph) versus a video clip

• Sequence - Folding - Structure - Dynamics

Lactobacillus casei dihydrofolate reducatase PDB id 2LF1

• http://image.shutterstock.com/display_pic_with_lo

go/84324/84324,1169286790,1/stock-photo-auto- mechanic-working-under-the-car-2517812.jpg http://mantivities.files.wordpress.com/ 2008/09/mechanic.jpg

Mechanics at work

• www.nature.com/nature/journal/v432/n7015/fig_tab/nature02981_F6.html

Nature (2004) 432:361

Conformational changes

• Unfolded

Folded PROTEIN

Human lysozyme PDB id 3FE0

?

Conformational changes

• PROTEIN

Nuclear co-activator binding domain of CREB-binding protein (CREB: cAMP response element binding) PDB id 2KKJ Sodium lauroyl sarcosinate-bound α α α α-synuclein PDB id 2KKW

Conformational changes

Unfolded Folded Partially folded states

• Bacillus subtilis yitJ S Box/SAM-I Riboswitch

PDB id 3NPB

?

Conformational changes

Unfolded Folded RNA

• 2SO-α

α α α-L-iduronopyranose

4C1-α

α α α-L-idopyranose

• CH2OH - axial
• OH - equatorial

1C4-α

α α α-L-idopyranose

• OH - axial
• CH2OH - equatorial

Conformational changes

• OH - equatorial
• COOH - equatorial

• Valley between two mountains, a water well?

• Curr. Opin. Struct. Biol. (2011) 21:426

Energy landscape of a protein

Folded protein Intrinsically disordered well defined “minimum energy” or “stable” state

• Ec

cc State MbCO Science (1991) 254:1598

Myoglobin (Mb): Mb, MbCO, or MbO2

Ec: potential energy cc: conformational coordinates

Sequence - Folding - Structure - Dynamics - Function

• ensemble: a group producing a single effect

Helicobacter pylori SlyD PDB id 2KR7

May contain “correctly” folded (inactive or active)

• r misfolded forms

May contain inactive or active forms

Conformational ensembles

Unfolded Folded PROTEIN

• Ec

Ec cc cc0 State Tier 0 MbCO A0 A1 A3

Ec: potential energy cc: conformational coordinates

MbCO: different states (low temp flash photolysis) Different states: stretching of bound CO different (IR) Rate of binding CO: different (A0, fastest; A3, slowest) Regulation by switching between different states Myoglobin (Mb): Mb, MbCO, or MbO2

Energy landscape of myoglobin Sequence - Folding - Structure - Dynamics - Function

• Ec: potential energy

cc: conformational coordinates

Science (1991) 254:1598

MbCO: different states (low temp flash photolysis) Different states: stretching of bound CO different (IR) Rate of binding CO: different (A0, fastest; A3, slowest) Regulation by switching between different states

Ec Ec Ec cc cc0 cc1 State Tier 0 Tier 1 MbCO A0 A1 A3

Myoglobin (Mb): Mb, MbCO, or MbO2 Different substates: IR and kinetics of re-binding after photodissociation

Sequence - Folding - Structure - Dynamics - Function

• Ec

Ec Ec Ec cc cc0 cc1 cc2 State Tier 0 Tier 1 Tier 2 MbCO A0 A1 A3

Ec: potential energy cc: conformational coordinates

Science (1991) 254:1598

MbCO: different states (low temp flash photolysis) Different states: stretching of bound CO different (IR) Rate of binding CO: different (A0, fastest; A3, slowest) Regulation by switching between different states Myoglobin (Mb): Mb, MbCO, or MbO2 Different substates: IR and kinetics of re-binding after photodissociation

Sequence - Folding - Structure - Dynamics - Function

• Concept of landscapes: molecular energy landscapes

Multi-dimensional landscapes - we, as humans, can not visualize them We need to formulate our interpretation / understanding / make testable hypothesis around “imaginary” landscapes Visualizing “natural landscapes” helps us in our imagination

• Figure 1a

Nature (2007) 450:964

Tier 0:

• Barrier height -- interconversion rate
• Population -- Boltzmann distribution (∆

∆ ∆ ∆G)

• Conformational substates – closely related

Tier 0:

• Depends on the energy landscape
• Landscape – environment, interactions
• Change in system – mutation

Regulation of function by altering energy landscapes

• Conformational substates

Nature (2011) 477:111

• Conformational substates

Figure 4d to 4f of Nature (2011) 477:111 T4 lysozyme Leu99Ala Leu99Ala, Gly113Ala double mutant Leu99Ala, Gly113Ala, Arg119Pro triple mutant

• Conformational substates
• Nat. Chem. Biol. (2011) 7:411

• 3D structure by x-ray crystallography

Incompatible with biochemical data. Trends Biochem. Sci. (2005) 30:166 see, also, Science (2005) 309_897 [2A79] “compatible” structure

Integrate data from biophysical, biochemical, structural, and computational studies

Only one of the four subunits is displayed here Nature (2003) 423:33 (1ORQ)

http://store.bioetch.com/ (http://www.youtube.com/watch?v=FRJfnhUImA0)

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• Timescale of dynamic processes in proteins
• µ

µ µ µ

• Local flexibility

Collective motions Side-chain rotamers Larger domain motions Loop motion Methyl rotation Bond vibration NMR relaxation Fluorescence Raman spectroscopy IR spectroscopy MD simulations (computational method) UV-Vis spectroscopy X-ray diffraction H-D exchange Timescale Method Nature

• f

motion Figure 1b Nature (2007) 450:964

• Molecular motions
• 1. Translation
• 2. Tumbling or rotation (ns; fluorescence)
• 3. “Breathing” (ns; H-D exchange)
• 4. Bond stretching and angle bending (fs; vibrational spectroscopy)
• 5. Side chain rotation (ns; NMR)
• 6. Loop, hinge, domain motions (ns to ms; fluorescence, NMR, x-ray)
• 7. Folding of proteins (ms to s; fluorescence, kinetic+equilibrium)

A Fersht (1999) Structure and mechanism in protein science. pp45-51 M B Jackson (2006) Molecular and cellular biophysics. pp98-104.

rigid body motions

• Energy landscape of a protein

Energy landscapes of a protein can be re-modelled i.e., the dynamics of the protein is altered: by its environment (solvent, pH, ...), by binding with a ligand (small molecule or macromolecule) The protein has different conformational sub-states. In response to the “environmental cues”, it samples different sub-states to different extents. This allows the protein to communicate to or interact with its surroundings. Without this capability, protein is essentially “dead” and is of no use to the organism.

Nature (2007) 450:964 Science (2009) 324:198

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Assembly or aggregation

! Turkey hemoglobin PDB id 3K8B /! Sperm whale myoglobin PDB id 2W6W

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Assembly or aggregation

• One protein, two conformations, two functions...
• J. Mol. Biol., (2006) 359:1045

Bcl-2 family of proteins: involved in the regulation of apoptosis Localization: (1) cytosol and (2) Mitochondrial outer membrane Conformations in the two locations are DIFFERENT Function in the two locations is also DIFFERENT cytosol: α α α α-helical bundles, integrates various cellular signals membrane: regulate the release of CytC and other apoptotic factors

• “Molecular” dynamics

Protein dynamics – better characterized vis-à-vis other biomolecules Multi-dimensional energy landscape describes probabilities of the conformational states (thermodynamics) energy barrier + transitions (kinetics) Paradigm shift structure – function (old) structure – dynamics – function (current)

Nature (2007) 450:964 Science (2009) 324:198

• Summary...

Small molecules can exist in different conformations Macromolecules can also exist in different conformations folded, unfolded, etc. (for monomers) aggregates or oligomers (homo- or hetero-) or assembly Molecules are dynamic, NOT static Natural landscapes and energy landscapes Conformational states, substates, ... Altering the energy landscapes of proteins role of solvent, co-solvent, co-solutes, ligand, ... Dynamics – rigid body + internal Time scales of dynamics Two aspects of dynamics: thermodynamics and kinetics