Bioch/BIMS 503 Lecture 2 Structure and Function of Proteins August 28, 2008 Robert Nakamoto rkn3c@virginia.edu 2-0279 Secondary Structure Φ Ψ angles determine protein structure Φ Ψ angles are restricted by steric constraints The α-helix - regular local H-bond pattern The α-helix has a dipole moment Other helices β-strands - parallel and anti-parallel β-turns The statistics of α-helices and β-sheets - objective definitions of α-helices/β-sheets Prediction of transmembrane α-helices Further Reading Φ, Ψ angles alone determine protein structure Lehninger Biochemistry, Chapter 4, pp. 116-125 Matthews and van Holde (MvH) Biochemistry, Chapter 6, pp. 161-170, 180-181. Branden and Tooze (BT), Introduction to Protein Structure, Chapter 2, pp. 13-22. Kabsch, W. and Sander, C. (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577-2637. Transmembrane Prediction Kyte, J. and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. Figure 6.2: Rotation around the bonds in a polypeptide chain. 1
φ,ψ angles are restricted by steric constraints Figure 6.8: A Ramachandran plot. Figure 6.9: A sterically nonallowed conformation. Figure 6.2: Rotation around the bonds in a polypeptide chain. Figure 6.9: A sterically nonallowed conformation. Figure 6.3: The α helix and β sheet. Figure 6.4: Other possible secondary structures of polypeptides. Regular (repeating) structures in proteins 2
The α-helix Other helical structures: π and 3,10 The α-helix has a dipole moment Many α-helices are amphipathic (hydrophilic on one side, hydrophobic on the other) BT Fig. 2.3 Negatively charged groups (P0 4 ) frequently bind to the amino-ends of α helices. The dipole moment of an α helix, as well as possible H-bonds to free NH groups at the end, favors such binding. Different helical faces can have different properties BT Fig. 2.4 The helical wheel. Amino acid residues are plotted every 100 o around the wheel. Green: hydrophobic; Blue: polar; Red: charged. The second helix is amphipathic. Eisenberg s Hydrophobic moment ( ) 2 + ( H i cos("i) ) 2 µ H = # H i sin("i) i " =100 o for $ - helix " =180 o for % - strand # i polar - red hydrophobic - green 3
β strands and β sheets Parallel and anti-parallel β sheets BT Fig 2.6 - parallel β sheet BT Fig 2.5 - antiparallel β sheet Figure 6.18: Examples of β turns. β-turns Figure 6.19: A γ turn. 4
Figure 6.10: Ramachandran plot of the residues in bovine pancreatic trypsin inhibitor (BPTI). Currently 10,340 protein fold families in Pfam [http://pfam.sanger.ac.uk/] Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile hidden Markov models. Generally does not include membrane proteins. Goals of the Protein Structure Initiative - Structural Genomics Results of structural genomics Efficient Protein Target Selection Gene Cloning and Expression Protein Production Crystal Production and Delivery Structure Determination and Refinement Model Validation and Data Dissemination Structure-based functional assignment is still a challenge 5
Statistics of secondary structures Frequency of secondary structures W. Kabsch and C. Sander (1983) Biopolymers 22:2577-2637 W. Kabsch and C. Sander (1983) Biopolymers 22:2577-2637 Transmembrane secondary structures Glycophorin: an example transmembrane protein Note the predominance of hydrophobic amino acids within the bilayer. There is a huge energy penalty to place a charge in the low dielectric milieu of the bilayer. Charged residues are often in the form of ion pairs. Positive charged residues are often found at the cytoplasmic surface ( Positive Inside Rule ) BT Fig. 12.1 Aromatic residues are often found just inside the bilayer Glycosylation only occurs on the luminal or extracellular side 6
Examples of different topologies of membrane proteins: Topology of the HelB, HelC, and HelD proteins Difficulties in determination of membrane protein structure Usually very low expression levels Must remove the protein from its native environment, i.e., solubilization in detergents must identify the best detergent for solubility and stability Relatively low stability, especially in detergents Relatively highly dynamic with multiple conformations Relatively little surface exposure for crystal contacts From Goldman et al, Proc. Natl. Acad. Sci. USA, 1998. 95:5003-5008 Ca 2+ ATPase Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues. Toyoshima, C., Nomura, H. and Tsuda, T. (2004). Nature 432, 361-368. Ion Channel Structure: the bacterial K + channel, KcsA 7
Porin Structure of porin refined at 1.8 Å resolution. Weiss MS, Schulz GE. J Mol Biol 1992, 227, 493-509 ph Sensitive conformational change exposes the Influenza virus hemagglutinin fusion peptide The exposed fusion peptide inserts into the target membrane leading to membrane fusion Amino acid Hydropathicity/Hydrophobicity Hopp T.P., Woods K.R. (1981) PNAS. 78:3824-3828. Kyte J., Doolittle R.F. (1982). J. Mol. Biol. 157:105-132 D. M. Engelman, T. A. Steitz, A. Goldman, (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321 Hopp/ Woods Arg: Lys: Asp: Glu: Ser: Gln: Asn: Pro: Gly: Thr: His: Ala: Cys: Met: Val: Leu: Ile: Tyr: Phe: Trp: 3.0 3.0 3.0 3.0 0.3 0.2 0.2 0.0 0.0-0.4-0.5-0.5-1.0-1.3-1.5-1.8-1.8-2.3-2.5-3.4 Kyte/ Doolittle Arg: Lys: Asp: Glu: Gln: Asn: His: Pro: Tyr: Trp: Ser: Thr: Gly: Ala: Met: Cys: Phe: Leu: Val: Ile: -4.5-3.9-3.5-3.5-3.5-3.5-3.2-1.6-1.3-0.9-0.8-0.7-0.4 1.8 1.9 2.5 2.8 3.8 4.2 4.5 GES Arg: Asp: Lys: Glu: Asn: Gln: His: Tyr: Pro: Ser: Gly: Thr: Ala: Trp: Cys: Val: Leu: Ile: Met: Phe: Prediction of membrane topology based on hydropathy analysis 12.3 9.2 8.8 8.2 4.8 4.1 3.0 0.7 0.2-0.6-1.0-1.2-1.6-1.9-2.0-2.6-2.8-3.1-3.4-3.7 8
R -4.5-4.5-4.5-4.5-4.5-4.5-4.5 K -3.9-3.9-3.9-3.9-3.9-3.9-3.9 D -3.5-3.5-3.5-3.5-3.5-3.5-3.5 B -3.5-3.5-3.5-3.5-3.5-3.5-3.5 N -3.5-3.5-3.5-3.5-3.5-3.5-3.5 S -0.9-0.9-0.9-0.9-0.9-0.9-0.9 E -3.5-3.5-3.5-3.5-3.5-3.5-3.5 H -3.2-3.2-3.2-3.2-3.2-3.2-3.2 Z -3.5-3.5-3.5-3.5-3.5-3.5-3.5 Q -3.5-3.5-3.5-3.5-3.5-3.5-3.5 T -0.7-0.7-0.7-0.7-0.7-0.7-0.7 G -0.4-0.4-0.4-0.4-0.4-0.4-0.4 A 1.8 1.8 1.8 1.8 1.8 1.8 1.8 P -1.6-1.6-1.6-1.6-1.6-1.6-1.6 V 4.2 4.2 4.2 4.2 4.2 4.2 4.2 Y -1.3-1.3-1.3-1.3-1.3-1.3-1.3 C 2.5 2.5 2.5 2.5 2.5 2.5 2.5 M 1.9 1.9 1.9 1.9 1.9 1.9 1.9 I 4.5 4.5 4.5 4.5 4.5 4.5 4.5 L 3.7 3.7 3.7 3.7 3.7 3.7 3.7 W -0.9-0.9-0.9-0.9-0.9-0.9-0.9 F 2.7 2.7 2.7 2.7 2.7 2.7 2.7 X 0.0 0.0 0.0 0.0 0.0 0.0 0.0 E V N N E S F V I Y M F V V H F T -8.0 0.0 7.6 18.9 13.0 11.8-0.3 2.2 13.8 20.4 11.2 R K D B N S E H Z Q T G A P V Y C M I L W F -4.5-4.5-4.5-4.5-4.5-4.5-4.5-4.5-4.5-3.9-3.9-3.9-3.9-3.9-3.9-3.9-3.9-3.9-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-0.9-0.9-0.9-0.9-0.9-0.9-0.9-0.9-0.9-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.2-3.2-3.2-3.2-3.2-3.2-3.2-3.2-3.2-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.5-3.7-3.7-3.7-3.7-3.7-3.7-3.7-3.7-3.7-0.7-0.7-0.7-0.7-0.7-0.7-0.7-0.7-0.7-0.4-0.4-0.4-0.4-0.4-0.4-0.4-0.4-0.4 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8-1.6-1.6-1.6-1.6-1.6-1.6-1.6-1.6-1.6 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2-1.3-1.3-1.3-1.3-1.3-1.3-1.3-1.3-1.3 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7-0.9-0.9-0.9-0.9-0.9-0.9-0.9-0.9-0.9 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 E V N N E S F V I Y M F V V H F T -8.0 0.0 7.6 18.9 13.0 11.8-0.3 2.2 13.8 20.4 11.2 R -4.5-4.5-4.5-4.5-4.5-4.5-4.5 K -3.9-3.9-3.9-3.9-3.9-3.9-3.9 D -3.5-3.5-3.5-3.5-3.5-3.5-3.5 B -3.5-3.5-3.5-3.5-3.5-3.5-3.5 N -3.5-3.5-3.5-3.5-3.5-3.5-3.5 S -0.9-0.9-0.9-0.9-0.9-0.9-0.9 E -3.5-3.5-3.5-3.5-3.5-3.5-3.5 H -3.2-3.2-3.2-3.2-3.2-3.2-3.2 Z -3.5-3.5-3.5-3.5-3.5-3.5-3.5 Q -3.5-3.5-3.5-3.5-3.5-3.5-3.5 T -0.7-0.7-0.7-0.7-0.7-0.7-0.7 G -0.4-0.4-0.4-0.4-0.4-0.4-0.4 A 1.8 1.8 1.8 1.8 1.8 1.8 1.8 P -1.6-1.6-1.6-1.6-1.6-1.6-1.6 V 4.2 4.2 4.2 4.2 4.2 4.2 4.2 Y -1.3-1.3-1.3-1.3-1.3-1.3-1.3 C 2.5 2.5 2.5 2.5 2.5 2.5 2.5 M 1.9 1.9 1.9 1.9 1.9 1.9 1.9 I 4.5 4.5 4.5 4.5 4.5 4.5 4.5 L 3.7 3.7 3.7 3.7 3.7 3.7 3.7 W -0.9-0.9-0.9-0.9-0.9-0.9-0.9 F 2.7 2.7 2.7 2.7 2.7 2.7 2.7 X 0.0 0.0 0.0 0.0 0.0 0.0 0.0 http://fasta.bioch.virginia.edu/fasta/grease.htm E V N N E S F V I Y M F V V H F T -8.0 0.0 7.6 18.9 13.0 11.8-0.3 2.2 13.8 20.4 11.2 9
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Secondary structure summary: Review questions The path of the Cα backbone (which determines the shape of the protein) is fully determined by the Φ and Ψ angles around each Cα Φ, Ψ values are strongly constrained by steric hindrance The two classes of repeating Φ,Ψ structures are helices (α, π, 3,10) and β strands (which make parallel and anti-parallel sheets) The interactions stabilizing α-helices and β-strands are backbone H-bonds (no role for side chain) α-helix H-bonds are local; β sheet interactions can be distant in sequence sequential β-strand side-chains face opposite directions Secondary structures in proteins are short; α-helices range from 4-20 amino acids (ave ~ 8-10); β-strands are shorter (ave ~ 4 amino acids) Transmembrane α-helices can be predicted from hydrophobicity 1. What is the direction of the α-helical dipole moment? 2. Why are δ=100 o and δ=180 o used to calculate Eisenberg s hydrophobic moment? 3. Order the following helical structures by helix diameter from smallest to largest? 4. Some proteins contain only β-strand secondary structure. What type of β-sheet must these strands form? 5. What is the minimum size of an α-helix? A β- strand? 6. What is the average size of an α-helix in a soluble protein? In a transmembrane domain? Questions from previous exams: 1. Describe the structural features exhibited by the polypeptide backbone of both types of beta-sheet. What is the effect of a single amino-acid insertion into a beta-strand? Why might one expect it to be more difficult to predict beta-strands than alpha-helices based on sequence information alone? Are beta-strands predicted less accurately? 2. Describe the structural features exhibited by the polypeptide backbone of an alpha-helix. Why does proline disrupt the regular pattern of an alpha-helix as proline is located in the middle of the helical segment, but has no disruptive effect when it is located at the N-terminus of the same helical segment? 3. Name two scales have been used for transmembrane helix prediction and describe how were they derived? Transmembrane helix prediction is very accurate; does this accuracy support the observation that most soluble proteins have a hydrophobic core? Why or why not? 4. Pick 5 amino acids, 2 hydrophobic, 2 charged, and 1 uncharged hydrophilic. (a) Name the 5 amino-acids, using their full name and either the standard 3-letter or 1-letter code. (b) Give each of the amino-acids an approximate hydropathy value, using a range of +2.. 2. (c) write down a 10-amino-acid sequence using your 5 amino-acids and plot a Kyte-Doolittle hydropathy plot using a 3-amino-acid window for the 10-amino-acid sequence. 11