Hydrophobic Residue Patterning in - Strands and Implications For - Sheet Nucleation

Hydrophobic Residue
Patterning in β-Strands and

Implications for β-Sheet
Nucleation
Brent Wathen
Dept. of Biochemistry
Queen’s University
Outline
• Part I: Introduction
• Proteins
• Protein Folding
• Part II: Protein Structure Prediction

• Goals, Challenges
• Techniques
• State of the Art
• Part III: Residue Patterning on β-Strands

• β-Sheet Nucleation
• Hydrophobic/Hydrophilic Patterning
2
Outline
• Proteins
• Protein Folding

• Techniques

3
Part I: Introduction
Proteins – Some Basics

• What Is a Protein?
4

• Linear Sequence of Amino Acids...
5

• What is an Amino Acid?
6

• What is an Amino Acid?
7

• How many types of Amino Acids?
8

• How many types of Amino Acids?
• 20 Naturally Occurring Amino Acids
• Differ only in SIDE CHAINS
Isoleucine Arginine Tyrosine
9

• Amino Acids connect via PEPTIDE BOND
10

• Backbone can swivel:
DIHEDRAL ANGLES
• 2 per Amino Acid
• Proteins can be 100’s of
Amino Acids in length!
• Lots of freedom of
movement
11
Protein Functions
• What do proteins do?
12
Protein Functions
• Enzymes
• Cellular Signaling
• Antibodies
13
Protein Functions
• Enzymes
• Antibodies
• WHAT DON’T THEY DO!
14
Protein Functions
• Enzymes
• Antibodies
• WHAT DON’T THEY DO!
• Comes from Greek Work Proteios – PRIMARY

• Fundamental to virtually all cellular processes
15
Protein Functions
• How do proteins do so much?
16
Protein Functions
• How do proteins do so much?
• Proteins FOLD spontaneously
• Assume a characteristic 3D SHAPE
• Shape depends on particular Amino Acid
Sequence
• Shape gives SPECIFIC function
17
Protein Structure
• STRUCTURE  FUNCTION relationship
• Determining structure is often critical in
understanding what a protein does
• 2 main techniques
• X-ray crystallography
• NMR
• 0.5Å RMSD accuracy
• Both are very challenging
• Months to years of work
• Many proteins don’t yield to these methods
18
Protein Structure
• Levels of organization
• Primary Sequence
• Secondary Structure (Modular building blocks)
• α-helices
• β-sheets
• Tertiary Structure
• Quartenary Structure
• Hydrophobic/Hydrophilic Organization
• Hydrophobics ON INSIDE
• Hydrophobic Cores
19
Protein Structure
20
Protein Structure
21
Protein Folding
• What we DO know...
• Protein folding is FAST!!
• Typically a couple of seconds
• Folding is CONSISTENT!!
• Involves weak forces – Non-Covalent
• Hydrogen Bonding, van der Waals, Salt Bridges
• Mostly, 2-STATE systems
• VERY FEW INTERMEDIATES
• Makes it hard to study – BLACK BOX
22
Protein Folding
• What we DON’T know...
• Mechanism...?
• Forces...?
• Relative contributions?
• Hydrophobic Force thought to be critical
23
Intro Summary
• Proteins are central to all living things
• Critical to all biological studies
• Folding process is largely unknown
• Sequence  Structure Mapping
• Structure  Function relationship
• Determining Protein Structure Experimentally is
HARD WORK
24
Outline
• Proteins
• Protein Folding

• Techniques

25
Part II: Structure Prediction
The Prediction Problem
Can we predict the final 3D protein structure

knowing only its amino acid sequence?
26
The Prediction Problem
Can we predict the final 3D protein structure

knowing only its amino acid sequence?
• Studied for 4 Decades

• “Holy Grail” in Biological Sciences
• Primary Motivation for Bioinformatics
• Based on this 1-to-1 Mapping of Sequence to
Structure
• Still very much an OPEN PROBLEM
27
PSP: Goals
• Accurate 3D structures. But not there yet.
• Good “guesses”
• Working models for researchers
• Understand the FOLDING PROCESS
• Get into the Black Box
• Only hope for some proteins
• 25% won’t crystallize, too big for NMR
• Best hope for novel protein engineering
• Drug design, etc.
28
PSP: Major Hurdles

• Energetics
• We don’t know all the forces involved in detail
• Too computationally expensive BY FAR!
• Conformational search impossibly large

• 100 a.a. protein, 2 moving dihedrals, 2 possible positions
for each diheral: 2200 conformations!
• Levinthal’s Paradox
• Longer than time of universe to search
• Proteins fold in a couple of seconds??
• Multiple-minima problem
29
Tertiary Structure Prediction

• Major Techniques
• Template Modeling
• Homology Modeling
• Threading
• Template-Free Modeling
• ab initio Methods
• Physics-Based
• Knowledge-Based
30
Template Modeling
• Homology Modeling
• Works with HOMOLOGS
• ~ 50% of new sequences have HOMOLOGS
• BLAST or PSI-BLAST search to find good models
• Refine:
• Molecular Dynamics
• Energy Minimization
31
Template-Free Modeling
• Modeling based primarily from sequence
• May also use: Secondary Structure Prediction,
analysis of residue contacts in PDB, etc.
• Advantages:
• Can give insights into FOLDING MECHANISMS
• Adaptable: Prions, Membrane, Natively Unfolded
• Doesn’t require homologs
• Only way to model NEW FOLDS
• Useful for de novo protein design
• Disadvantages: HARD!
32
• Physics-Based
• Use ONLY the PRIMARY SEQUENCE
• Try to model ALL FORCES
• EXTREMELY EXPENSIVE computationally
• Knowledge-Based
• Include other knowledge: SSP, PDB Analysis
• Statistical Energy Potentials
• Not so interested in folding process
• “Hot” area of research
33
• All methods SIMPLIFY problem
• Reduced Atomic Representations
• C-α’s only; C-α + C-β; etc.
• Simplify Force Fields
• Only van der Waals; only 2-body interactions
• Reduced Conformational Searches
• Lattice Models
• Dihedral Angle Restrictions
34
• Basic Approach:
1. Begin with an unfolded conformation
2. Make small conformational change
3. Measure energy of new conformation
Accept based on heuristic: SA, MC, etc.
4. Repeat until ending criteria reached
• Underlying Assumption:
Correct Conformation has LOWEST ENERGY
35
Diverse Efforts
• Data Mining
• Pattern Classification
• Neural Networks, HMMs, Nearest Neighbour, etc.
• Packing Algorithms
• Search Optimization
• Traveling Salesman Problem
• Contact Maps, Contact Order
• Constraint Logic, etc.
• Combinations of the above!
36
ROSETTA
• Pioneered by Baker Group (U. of Washington)
• Fragment Based Method
• Guiding Assumption:
• Fragment Conformations in PDB approximate their
structural preferences
• Pre-build fragment library
• Alleviates need to do local energy calculations
• Lowest energy conformations should already be in
library
37
ROSETTA
• Pre-build fragment library
• 3-mers and 9-mers
• 200 structural possibilities for each
• Build conformations from the library
• Randomly assign 3-mers, 9-mers along chain
• During conformational search, reassign a 3-mer or a
9-mer to a new conformation at random
• Score using energy function
• Adaptive: Coarse grain at first, detailed at end
• Accept changes based on Monte Carlo method
38
Diverse Efforts
• Data Mining
• Pattern Classification
• Neural Networks, HMMs, Nearest Neighbour, etc.
• Packing Algorithms
• Search Optimization
• Traveling Salesman Problem
• Contact Maps, Contact Order
• Constraint Logic, etc.
• Combinations of the above!
39
State of the Art

• CASP Competition
• Critical Assessment of Structure Prediction
• Blind Competition Every 2 years
• CASP6 in 2004 - CASP7 just completed
• ~75 proteins whose structures have not been
published as yet
• Easy homologs examples
• Distant homologs available
• De novo structures: no homologs known
40
State of the Art

CASP6 Target 266

(green), and best
model (blue)
Moult, J. (2005) Cur. Opin.

Struct. Bio. 15:285-289
41
State of the Art

• Alignment still not easy, and often requires multiple
templates
• Accurate core models (within 2-3Å RMSD)
• Still not good at modeling regions missing from
template
• Side-chain modeling not too good
• Molecular dynamics not able to improve models as
hoped
42
State of the Art

CASP6
target 201,
and best
model.
Vincent, J.J. et. al (2005)
Proteins 7:67-83.
43
State of the Art

CASP6 target
241, and 3 best
models.

Proteins 7:67-83.
44
State of the Art

• How Good are Current Techniques?
• CASP6 Summary:
“The disappointing results for [hard new fold] targets
suggest that the prediction community as a whole
has learned to copy well but has not really learned
how proteins fold.”
Proteins 7:67-83.
45
PSP Summary
• Many diverse, creative efforts
• Progress IS being made in finding final 3D
structures
• Less so with regards to understanding folding
mechanisms
• NEEDED:
• Marriage of Creative Ideas and Increased
Resources
46
Outline
• Proteins
• Protein Folding

• Techniques

47
Part III: β-Strand Patterning
β-Sheet Basics
• Made up of β-Strands
• Diverse:
• Parallel/Antiparallel
• Edge/Interior Strands
• Typically Twisted
• Many Forms
• β-sandwiches, β-barrels, β-helices, β-propellers, etc.
• 2D? 3D?
• Less studied than helices
48
Beta Sheet Basics
Internalin A Narbonin
Polygalacturonase
Galactose Oxidase
49
Beta Sheet Basics

• What do we know?
 Residues:
• V, I, F, Y, W, T, C L
• Found largely in Protein Cores
• Amphipathic Nature
50
Amphipathic
51
Theory of β-Sheet Nucleation

• Hydrophobic Zipper (HZ)
• Dill et. al. (1993)
• Hydrophobic residues from different parts of
chain make initial contact
• Correct alignment of backbones
• Hydrogen bonding
• Subsequent growth via “Zipping Up”
52
Theory of β-Sheet Nucleation

Dill, K.A. et al., (1993)

Proc. Natl. Acad. Sci.
USA 90: 1942-1946.
53
Theory of Nucleation
• Once Hydrophobic “Seed” established, can
grow out 2 directions
54
Thought Experiment...
• What would a Beta Seed look like?
55
• Contain hydrophobics
• On both strands
56
• On both strands
• How many?
• Will single hydrophobic on each strand be
sufficient?
57
• On both strands
• How many?
sufficient?
• Single Unlikely:
• 1 Hydrophobic Residue NOT SPECIFIC ENOUGH
• Too many possible combinations
58
• On both strands
• How many?
sufficient?
• Single Unlikely:
• 1 Hydrophobic Residue NOT SPECIFIC ENOUGH
• Too many possible combinations
At least 1 strand must have >1 Hydrophobic

59
• What hydrophobic arrangement would lead to
Beta Sheet Nucleation?
• i,i+1?
• i,i+2?
• i,i+3?
• i,i+4?
60
• i,i+1? No, not likely: Amphipathic.
• i,i+2?
• i,i+3?
• i,i+4?
61
• i,i+2?
• i,i+3? No... Amphipathic.
• i,i+4?
62
• i,i+2?
• i,i+4? Seems too far apart...
63
• i,i+2? Most likely.
• i,i+4? Seems too far apart... Chain loop?
64
Hypothesis
Assuming:
• Beta Sheets Nucleate by Hydrophobics (HZ)
• i,i+2 hydrophobic pairings on beta strands are
necessary for nucleation
65
Hypothesis
Assuming:
• Sec. structures contain their nucleating residues
• Beta Sheets Nucleate by Hydrophobics (HZ)
• i,i+2 hydrophobic pairings on beta strands are
necessary for nucleation
Beta Strands contain an increased frequency of

i,i+2 hydrophobic residue pairings.
66
Hypothesis
67
Hypothesis
68
Hypothesis
69
Hypothesis
70
Technique
• Looking for statistically significant patterns
• For any particular pattern:
1. Count how often it occurs in database
2. Randomly shuffle residues in sheets
3. Re-count how often pattern occurs
4. Repeat random shuffle and counting x1000
5. Compare initial count, avg random count
Calculate the Std Dev σ
If σ > 3.0, statistically significant
71
Technique
72
Technique
73
Technique
74
Technique
75
Technique
76
Technique
77
Technique
• Patterns of Interest:
• Hydrophobic patterning (V L I F M)
• Hydrophilic patterning (K R E D S T N Q)
• Positions:
• i,i+1
• i,i+2
• i,i+3
• i,i+4
• Consider only strands of length >= 5 residues
78
Results
• Hydrophilics
• i,i+1
79
Results
• Hydrophilics
• i,i+1
• Strongly Disfavoured: -20.5σ

80
Results
• Hydrophilics
• i,i+2
81
Results
• Hydrophilics
• i,i+2
• Strongly Favoured: 13.0σ

82
Results
• Hydrophilics
• i,i+3
83
Results
• Hydrophilics
• i,i+3

84
Results
• Hydrophilics
• i,i+4
85
Results
• Hydrophilics
• i,i+4
• Strongly Favoured: 5.7σ

86
Results
• Hydrophilics: Summary
15
10
5
z- 0
Score -5
-10 (i,i+1) (i,i+2) (i,i+3) (i,i+4)
-15
-20
-25
Pattern
• Demonstrate Amphipathic Separation

• Suggests residues help guide tertiary formation
• Moral Support: Technique seems sound
87
Results
• Hydrophobics
• i,i+1
88
Results
• Hydrophobics
• i,i+1

89
Results
• Hydrophobics
• i,i+3
90
Results
• Hydrophobics
• i,i+3

91
Results
• Hydrophobics
• i,i+2
92
Results
• Hydrophobics
• i,i+2
• Barely Favoured!: 3.5σ

93
Results
• Hydrophobics
• i,i+4
94
Results
• Hydrophobics
• i,i+4

95
Results
• Hydrophobics: Summary
5
0
(i,i+1) (i,i+2) (i,i+3) (i,i+4)
-5
z-
-10
Score
-15
-20
-25
Pattern
• Clearly amphipathic: i,i+1 i,i+3 Disfavoured

• NOT particularly favoured at i,i+2 
• Unexpectedly: i,i+4 strongly Disfavoured
96
Results
• Hydrophobics: Summary
• Where are the hydrophobic pairings??
• Not at i,i+1 or i,i+3 or i,i+4
• Barely at i,i+2
• Note:
• Moderate i,i+2 pairing: No strong aggregation
• Low low i,i+4 pairing: Not Dispersed! Isolated
97
Results
98
Results
99
Results
• Examine localized hydrophobic pairings...
100
Results
• i,i+2 @ NT
101
Results
• i,i+2 @ NT
• Only slightly favoured: 2.5σ
102
Results
• i,i+2 @ NT+1
103
Results
• i,i+2 @ NT+1
• Strongly favoured!!: 9.3σ
104
Results
• i,i+2 @ NT+2
105
Results
• i,i+2 @ NT+2
• Indifferent: 0.8σ
106
Results
• i,i+2 @ CT
107
Results
• i,i+2 @ CT
• Favoured!: 5.7σ
108
Results
• i,i+2 @ CT-1
109
Results
• i,i+2 @ CT-1
110
Results
• i,i+2 @ CT-2
111
Results
• i,i+2 @ CT-2
112
Results
• i,i+2 @ Interior Positions
113
Results
• i,i+2 @ Interior Positions
• Actually Disfavoured!!: -3.0σ
114
Results
• Summary:
10
z- 4
Score 2
0
NT NT+1 NT+2 Central CT-2 CT-1 CT Avg
-2
-4
Pattern Location
• Localized i,i+2 hydrophobic pairing at NT and CT

• Disfavoured at interior positions
115
Results
• Are these patterns sense-specific?
• @ NT+1:
10
z- 4
Score 2
0
Parallel Antiparallel Mixed Edge
-2
-4
Strand Type
• Favoured for Parallel, Antiparallel

116
Results
• Are these patterns sense-specific?
• @ CT:
5
3
z-
2
Score
1
0
Parallel Antiparallel Mixed Edge
-1
Strand Type
• Favoured for Antiparallel, Mixed

• NOT PARALLEL!
117
Conclusions
• Hydrophobic patterning suggests:
• Hydrophobics are located on one side of beta
sheets  AMPHIPATHIC
• Hydrophobics are CLUSTERED
• Hydrophobics aggregate at NT, CT
• Parallel Strands: @ NT only
• Antiparallel Strands: @ NT & CT
• Supports HYDROPHOBIC ZIPPER theory for

sheet nucleation
118
Implications
• How do beta sheets nucleate?
• Parallel
119
Implications
• Parallel
• Nucleate at NT
• Growth is unidirectional: NTCT
120
Implications
• Antiparallel
121
Implications
• Antiparallel
• Nucleate at edge
• Growth is unidirectional
122
Future Work
1. Extend this work to 2D
Both intra- and inter-strand patterning
2. Consider more complex patterning
3 residues on one strand? NT Position?
Specific residue combinations?
3. Consider patterning by beta-sheet type
Beta Helices, Barrels, Sandwiches, etc.
123
Acknowledgements
• Dr. Jia
• Lab Members
• • Andrew Wong
Dr. Qilu Ye
• Michael Suits
• Dr. Vinay Singh
• Laura van Staalduinen
• Dr. Susan Yates
• Mark Currie
• Daniel Lee • Kateryna Podzelinska
• Jimmy Zheng
• Neilin Jaffer
• NSERC
124

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Hydrophobic Residue

Patterning in β-Strands and

• Part II: Protein Structure Prediction

• Part III: Residue Patterning on β-Strands

• Part II: Protein Structure Prediction

• Part III: Residue Patterning on β-Strands

Proteins – Some Basics

Proteins – Some Basics

Proteins – Some Basics

Proteins – Some Basics

Proteins – Some Basics

Proteins – Some Basics

Isoleucine Arginine Tyrosine

Proteins – Some Basics

Proteins – Some Basics

• Comes from Greek Work Proteios – PRIMARY

• Part II: Protein Structure Prediction

• Part III: Residue Patterning on β-Strands

The Prediction Problem

Can we predict the final 3D protein structure

The Prediction Problem

Can we predict the final 3D protein structure

• Studied for 4 Decades

PSP: Major Hurdles

• Conformational search impossibly large

Tertiary Structure Prediction

State of the Art

State of the Art

CASP6 Target 266

Moult, J. (2005) Cur. Opin.

State of the Art

State of the Art

State of the Art

Vincent, J.J. et. al (2005)

State of the Art

• Part II: Protein Structure Prediction

• Part III: Residue Patterning on β-Strands

Beta Sheet Basics

Beta Sheet Basics

Theory of β-Sheet Nucleation

Theory of β-Sheet Nucleation

Dill, K.A. et al., (1993)

At least 1 strand must have >1 Hydrophobic

Beta Strands contain an increased frequency of

• Consider only strands of length >= 5 residues

• Strongly Disfavoured: -20.5σ

• Strongly Favoured: 13.0σ

• Strongly Disfavoured: -6.1σ

• Strongly Favoured: 5.7σ

• Demonstrate Amphipathic Separation

• Strongly Disfavoured: -16.8σ

• Strongly Disfavoured: -16.6σ

• Barely Favoured!: 3.5σ

• Strongly Disfavoured: -19.6σ

• Clearly amphipathic: i,i+1 i,i+3 Disfavoured

• Only slightly favoured: 2.5σ

• Strongly favoured!!: 9.3σ

• Only slightly favoured: 3.4σ

• Only slightly favoured: 3.9σ

• Actually Disfavoured!!: -3.0σ

• Localized i,i+2 hydrophobic pairing at NT and CT

• Favoured for Parallel, Antiparallel