ProteinFoldingUniversityofNottingham：蛋白质折叠诺丁汉大学

N exp( Ei / kT ) Ni q q = partition function; N total number
Occupancy (Ni) of level i
Partition function
q is the sum of Boltzmann factors Reflects the number of thermally accessible states at the temperature of interest
Boltzmann distribution
Boltzmann distribution
Distribution of conformations over the available energy levels. Which is the most probable? Boltzmann distribution is the outcome of blind chance occupation of energy levels, subject to the requirement that the total energy has a particular value
Anfinsen Experiment
• After renaturation, the refolded protein has native activity, despite 105 ways to renature the protein. • Conclusion: All the information necessary for folding into its native structure is contained in the amino acid sequence of the protein.
• Many proteins form molten globules at low pH
• Model for early stages of protein folding (hydrophobic collapse)
The Folding Funnel
• A new view of protein folding suggested that there is no single route, but a large enseny dimensional funnel to its native structure. • Progress from the top to the bottom of the funnel is accompanied by an increase in the native-like structure as folding proceeds.
individual secondary structure elements
Nuclear condensation model
N
C
Supported by protein engineering studies and various theoretical calculations
Nuclear condensation model
Levinthal & Landscapes
• Structure space 3100 conformations • Sequence space 20100 sequences
Figure from Englander & co-workers, Proc Natl Acad Sci 98 19104 (2001)
EHH = -e
1 Prob (Nat)
DG = -kT {Prob(Nat)/Prob(Unf)}
UNFOLDED 0 Tm T
Folding landscapes and the Levinthal paradox
Flat landscape (Levinthal paradox)
Tunnel landscape (discrete pathways)
Anfinsen Experiment
• Remove b-mercaptoethanol only, oxidation of the sulfhydryl group, then remove urea → scrambled protein, no activity • Further addition of trace amounts of b-mercaptoethanol converts the scrambled form into native form. • Conclusion: The native form of a protein has the thermodynamically most stable structure.
N
C
Formation of a nucleus of hydrophobic residues
Nuclear condensation model
N C
Expansion of nucleus
Framework and Condensation models are extremes on a continuum
Molten Globule State
• Collapsed, with native-like 2ºstructure (far UV CD) • Weak or transient side-chain interactions (near UV CD) • Binds hydrophobic dyes
5. Biomolecular Modelling 6. Bioinformatics
Protein Folding
• Protein folding considers the question of how the process of protein folding occurs, i. e. unfolded native state. • This very challenging problem has been described as the second half of the genetic code, and as the three-dimensional code, as opposed to the one-dimensional code involved in nucleotide/amino acid sequence. • Importance: – Predict 3D structure from primary sequence – Avoid misfolding related to human diseases – Design proteins with novel functions
Models of Protein Folding
Framework model of protein folding
N
C
Supported by experimental observation of rapid formation of secondary structure during protein folding process
q Boltzm ann factors q exp( Ei / kT )
i
Toy protein model
Red – Hydrophobic (H) Black – Polar (P) HP model 1 conformation: E = -e
4 conformations: E = 0
Partition function – toy model
Q = 4 exp(-E0/kT) + exp(-E1/kT) Let E0 = 0 and E1 = -e, then Q = 4 + exp(e/kT) Prob (Native state), P = qNative/Q P = exp(e/kT)/{4 + exp(e/kT)}
Let us assume that protein can explore new conformations at the
same rate that bonds can reorient (1013 structures/second). Thus, the time to explore all of conformational space = 5 x 1047/1013 = 5 x 1034 seconds = 1.6 x 1027 years >> age of universe This is known as the Levinthal paradox.
The Levinthal paradox
Many proteins fold in seconds or less: how is this possible? Cyrus Levinthal tried to estimate how long it would take a protein to do a random search of conformational space for the native fold. Imagine a 100-residue protein with three possible conformations per residue. Thus, the number of possible folds = 3100 = 5 x 1047.
Framework model of protein folding
C
N
Formation of individual secondary structure elements
Framework model of protein folding
N
C
Coalescence and rearrangement of
Anfinsen Experiment
• Denaturation of ribonuclease A (4 disulfide bonds), with 8 M Urea containing bmercaptoethanol, leads to random coil and no activity
Protein Folding & Biospectroscopy Lecture 4
F14PFB David Robinson
Protein Folding
1. Introduction 2. Protein Structure 3. Interactions
4. Protein Folding Models
Realistic landscape (“folding funnel”)
Framework (or diffusion-collision): 2º structure forms independently and “dock” to form 3º structure Nucleation condensation: Concerted consolidation of 2º and 3º structure as nucleus expands Condensation when 2º structure inherently unstable in absence of 3º Structure. Framework becomes more probable as 2º elements become more stable