MCB65. Physical Biochemistry: Understanding Macromolecular Machines. Rachelle Gaudet Martin Samuels MCB 65 1/25/16 1

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1 MCB 65 Physical Biochemistry: Understanding Macromolecular Machines Rachelle Gaudet Martin Samuels 1/25/16 1

2 What do we need to know to understand macromolecular machines? 1/25/16 2

3 What do we need to know to understand macromolecular machines? 1/25/16 3

4 structure energetics structural methods energetics 1/25/16 4

5 energetics folding ligand binding protein-protein interactions reactions diffusion membranes metabolism regulatory networks 1/25/16 5

6 Teaching staff Instructors: Rachelle Gaudet Office hours: Northwest Labs Tuesdays 6 7PM Preceptor Martin Samuels (msamuels@fas.harvard.edu) Office hours: Fairchild 195 Fridays 3 5 PM Teaching Fellows: Enrique Garcia Rivera (egarciarivera@fas.harvard.edu) Office hours: Fridays 11 AM 12 PM Emily Ricq (elricq@fas.harvard.edu) Office hours: Mondays 11 AM 12 PM 1/25/16 6

7 Course Objective Become familiar and conversant in the physical properties and behaviors of biological molecules, with an emphasis on macromolecules Three dimensional structure and organization Energy classical & statistical thermodynamics Equilibria driving forces for reactions Kinetics of reactions Integration of the concepts 1/25/16 7

8 Lectures: Logistics Mondays, Wednesday and Fridays 10:00am 11:00am in NWL B 109 Sections: One mandatory combined discussion/laboratory session per week Monday noon 2:30PM or 6 8:30PM in NW room B 133 Must make ONE of these times to be able to take the class Prerequisites: LPSA or LS 1a, Chem 20 or 17, and Math 1b 1/25/16 8

9 Transporter associated with antigen processing (TAP) Hydrolysisdependent NBD opening peptide ER lumen TAP1 TAP2 ATP ATP ATP ATP cytoplasm ATP dependent NBD closure 1/25/16 9

10 MCB 65 Lab and Section Week Lab topic no section Journal club: TAP NBD structure paper PyMOL tutorial/selecting TAP NBD mutations No section (Presidents Day) Protein purification/ review for exam on the 25th SDS PAGE analysis of protein purification Dialysis No section (spring break) Bradford assay Protein stability assay Midterm exam review ATPase assay I ATPase assay II Final exam review Final lab report due (reading period) 1/25/16 10

PyMOL: Visualizing, analyzing and presenting macromolecular structures Through the semester, you will learn to use PyMOL to examine and present structures

11 PyMOL: Visualizing, analyzing and presenting macromolecular structures Through the semester, you will learn to use PyMOL to examine and present structures Tutorial in Section 2 Recurring presence in LPSs PyMOL files used in lectures available on Canvas Additional PyMOL resources available on the LPS page 1/25/16 11

12 Assignments and Grading Pop questions (10 x0.5): 5% 12 handed out in class 10 best out of 12 count Due at the beginning of the following lecture Lab and Problem Sets (10 x2): 20% Due on Wednesdays at the beginning of lecture, or before EXCEPT LPS1 which is due next Monday First LPS due Final lab report 10% a week from today! Midterms (1 x x 16): 35% Monday quiz on amino acids and nucleotides Wednesday covers lectures 1 12 Wednesday covers lectures Final: 30% During Finals period at 2pm Cumulative, emphasis final 1/3 of material 1/25/16 12

13 MCB 65 Collaboration Policy Discussion and the exchange of ideas are essential to doing academic work. We have designed a range of assignments and exercises for this course to both enhance and test your knowledge, and each has a different collaboration policy. LPS Assignments, Final Lab Report, and Pop Questions: In this course, you are encouraged to consult with your classmates as you work on assignments. However, after discussions with peers, make sure that you can work through the problem yourself and ensure that any answers you submit for evaluation are in your own words and the result of your own efforts. In addition, you must cite books, articles, websites, lectures, etc that have helped you with your work using appropriate citation practices. For more information on citation, you can consult the Guide to Citing in the Life Sciences Midterm and Final Exams: The Midterm Exams will occur during regular class periods, and the Final Exam during the exam period. All exams will be closed book and individual work the work should be all your own. 1/25/16 13

14 Reading materials Required text: The Molecules of Life: Physical and Chemical Principles by John Kuriyan, Boyana Komforti and David Wemmer 1/25/16 14

15 Reading materials II Supplemented with selected literature reviews Some optional, some required reading as detailed on page 4 of the syllabus All PDFs available on the course website Supplemental textbooks available on reserve at Cabot (listed on the syllabus and on the website) Contain many problems/exercises useful for additional practice 1/25/16 15

16 Learning the MCB 65 material Best practice for learning the material Read or at least skim ahead of the lecture Keep up to date with the readings Practice by working through problems problem sets, sections, additional problems (previous years LPSs and exams, end of chapter problems, other textbooks) Ask questions!! Take advantage of office hours! General guidelines for coursework: in college, expect ~3hrs of work on your own for each 1hr of lecture 1/25/16 16

17 Questions? 1/25/16 17

18 Lecture 1: Space, energy and time Reading: Lecture 1, today: Chapter 1, section A Lecture 2, Wednesday: Chapter 1: Supplemental reading: Chapters 1 and 2 provide a good review for materials that should already be familiar 1/25/16 18

19 Fundamental goal of physical biochemistry Understand and predict how macromolecules perform their biological function Requires knowledge of the physical principles that underlie macromolecular structure, interaction and function Develop a sense of scale for molecules and their transactions Three dimensional structure Classical & statistical thermodynamics Chemical kinetics Learning goals Space Energy Time Identify the different noncovalent interactions important for macromolecules 1/25/16 19

20 Macromolecule structure provides a spatial context to understand their function DNA polymerase in action Macromolecules are 3D objects of defined structure Noncovalent interactions control Structure Conformational states Interactions Lorena Beese, Duke University 1/25/16 20

21 Macromolecules are polymers of basic chemical building blocks RNA, DNA and proteins are the three major types of macromolecules They are sequences of monomers that are linked into polymers This sequence determines the macromolecule s informational and operational capabilities DNA: sequence encodes information Coding sequence Binding sites for transcriptional regulators Protein and RNA: sequence encodes 3D structure Folded entities perform biological reactions 1/25/16 21

to: tyrosine kinase Src Y 416 Y 527 Myr SH3 SH2 Kinase However,

22 There are multiple levels of protein structure Molecular biologists will often refer to the structure of the protein and show a diagram similar to: tyrosine kinase Src Y 416 Y 527 Myr SH3 SH2 Kinase However, biochemists and structural biologists have a different picture in mind: 1/25/16 22

genetic code, of the gene s DNA sequence Secondary structure Local conformation (i.e. noncovalent structure) of a

23 The levels of protein structure Primary structure Linear covalent chain of amino acids (from the 20 naturally available amino acids) Chemical structure of the protein chain Corresponds to the linear translation, using the genetic code, of the gene s DNA sequence Secondary structure Local conformation (i.e. noncovalent structure) of a polypeptide chain stabilized by hydrogen bonds Building blocks of the three dimensional protein structure helix, sheet, loop 1/25/16 23

structure Protein protein interactions leading to noncovalent complexes of at least two polypeptide chains

24 The levels of protein structure Tertiary structure The folding of secondary structure elements into a defined three dimensional arrangement Corresponds to the protein s threedimensional structure Quaternary structure Protein protein interactions leading to noncovalent complexes of at least two polypeptide chains Corresponds to the three dimensional arrangement of multiple subunits into a functional biological unit 1/25/16 24

25 Four levels of protein structure organization PyMOL representations 1/25/16 25

Most atom types have standard colors Carbon (variable color) Nitrogen Oxygen Sulfur Phosphate or Phosphate (often omitted) Molecular representations: Sticks show details of a

26 Most atom types have standard colors Carbon (variable color) Nitrogen Oxygen Sulfur Phosphate or Phosphate (often omitted) Molecular representations: Sticks show details of a structure Cartoon or ribbon diagram shows secondary structure Spheres details and packing, especially for small ligands Surfaces what other molecules can interact with 1/25/16 26

Packing inside proteins is very efficient Close packing of spheres: 3 2 0.

27 Packing inside proteins is very efficient Close packing of spheres: ~74% of volume occupied by atoms Organic liquids: ~45% of volume occupied by atoms Water: ~36% of volume occupied by atoms Proteins: ~70 75% How do they do it? Complex puzzle! 1/25/16 27

28 Proteins have a defined structure 1/25/16 28

Quote of note John Kendrew, on the structure of myoglobin, which he solved in 1958: Perhaps the most remarkable features of the molecule are its complexity and its lack of symmetry.

29 Quote of note John Kendrew, on the structure of myoglobin, which he solved in 1958: Perhaps the most remarkable features of the molecule are its complexity and its lack of symmetry. The arrangement seems to be almost totally lacking in the kind of regularities which one instinctively anticipates, and it is more complicated than has been predicted by any theory of protein structure Figure from The Molecules of Life ( Garland Science 2008) 1/25/16 29

Noncovalent forces shape structure Covalent interactions Chemical or primary structure Noncovalent interactions van der Waals interactions Hydrogen bonds Ionic interactions (salt

30 Noncovalent forces shape structure Covalent interactions Chemical or primary structure Noncovalent interactions van der Waals interactions Hydrogen bonds Ionic interactions (salt bridges) *** Both with self (intramolecular) and with surrounding solvent (intermolecular) Hydrophobic effect Figure from The Molecules of Life ( Garland Science 2008) 1/25/16 30

31 van der Waals interactions All atoms can form van der Waals interactions Weakest of noncovalent interactions Over very short distances ~3 4 Å Induced dipoles: What does 1 Å correspond to? Figure from The Molecules of Life ( Garland Science 2008) 1/25/16 31

Hydrogen bonds Dipoles partial charges Hydrogen bond between Aligned dipoles ~2.4 3.

32 Hydrogen bonds Dipoles partial charges Hydrogen bond between Aligned dipoles ~ Å Dipole is described as a vector between the two charges held apart, going from the negative charge to the positive charge. Figure from The Molecules of Life ( Garland Science 2008) 1/25/16 32

Strongest noncovalent interaction Salt bridges (Ion pairs) Electrostatic interaction is long range (compared to van der Waals and hydrogen bonds with short

33 Strongest noncovalent interaction Salt bridges (Ion pairs) Electrostatic interaction is long range (compared to van der Waals and hydrogen bonds with short effective distances) Salt bridge is an electrostatic interaction with the charged atoms close together Figure from The Molecules of Life ( Garland Science 2008) 1/25/16 33

34 Noncovalent interactions van der Waals ~ 1 2 kj/mol 1/25/16 34

35 Noncovalent interactions van der Waals ~ 1 2 kj/mol Ion pair ~ x vdw ~10 30 kj/mol depending on environment 1/25/16 35

36 Noncovalent interactions van der Waals ~ 1 2 kj/mol Ion pair ~ x vdw ~10 30 kj/mol depending on environment Hydrogen bond ~10 20 xvdw Less (~5 kj/mol) in H 2 0 1/25/16 36

37 Noncovalent interactions are readily made and broken at physiological temperatures Increasing energy (E) ~1000 kj/mol ~10 30 kj/mol ~5 kj/mol ~1 2 kj/mol Covalent bonds Noncovalent interactions ~ k B T~ Energy available from collisions and vibrations at physiological temperatures 1/25/16 37

The hydrophobic effect drives folding The hydrophobic effect is a collective property of solvent and solute molecules Hydrophobic molecules tend to cluster together to minimize their surface area

38 The hydrophobic effect drives folding The hydrophobic effect is a collective property of solvent and solute molecules Hydrophobic molecules tend to cluster together to minimize their surface area exposed to water Maximize the favorable interactions of water and hydrophilic molecules together Most important determinant of protein structure Figure from The Molecules of Life ( Garland Science 2008) 1/25/16 38

39 F0F1 ATP synthase runs at 6000 rpm Credits: John Walker 1/25/16 39

40 F 0 F 1 ATP synthase ~90 Å or 9 nm Whole assembly is ~0.5 MDa Turnover rate ~300 ATP / s ~100 revolutions / s ~3 revolutions / ms Each ATP stores ~50 kj/mol 1/25/16 40

41 Some concepts to remember Protein structures have a hierarchy Noncovalent interactions are crucial to macromolecule structure and interactions and range in energy van der Waals Hydrogen bond Ion pair/salt bridge Macromolecular functions are spread over a wide range of time scales 1/25/16 41

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