REVIEW
`
`The molten
`KUNIHIRO
`Department
`
`state
`
`globule
`KUWAJIMA’
`of Physics, School of Science, University
`
`of a-lactalhunun
`
`of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
`
`has made
`intermediates
`the folding
`of
`Our understanding
`20
`years.
`Intermediates
`the
`last
`during
`great
`advances
`a few proteins
`20 years
`in only
`that were
`characterized
`exceptional
`(13,
`14).
`as
`rather
`ago
`(12) were
`regarded
`structure
`of the folding
`on the
`Now we find many
`reports
`intermediates
`even
`at a level
`of atomic
`resolution.
`The
`availability
`of new experimental
`techniques
`was
`responsi-
`ble for
`this
`advance
`in protein
`folding
`studies.
`Studies
`by
`stopped-flow
`circular
`dichroism
`(CD)2
`techniques
`have
`unequivocally
`shown
`the presence
`of kinetic
`refolding
`in-
`termediates,
`which
`have
`appreciable
`amounts
`of secon-
`dary
`structure
`and
`accumulate
`within
`10 ms
`after
`the
`refolding
`starts
`in a globular
`protein
`(7, 15, 16). Hydro-
`gen-exchange
`labeling
`combined
`with 2D NMR spectros-
`copy has provided
`a powerful
`technique
`by which we can
`of
`characterize
`the
`structure
`refolding
`intermediates
`at
`the level of amino
`acid
`residue
`resolution
`(6, 17).
`inter-
`Many excellent
`reviews
`have
`appeared
`on folding
`mediates
`of globular
`proteins
`characterized
`by the
`ad-
`vanced
`techniques
`mentioned
`above(6,
`7, 15-21).
`This
`review therefore
`not attempt
`to be so comprehensive
`does
`as to include
`all aspects
`of kinetic
`folding
`intermediates.
`of a-
`It confines
`the
`subject
`to the molten
`globule
`state
`lactalbumin
`because
`this
`intermediate
`has
`been
`studied
`intensively
`in the
`last
`few years.
`a-Lactalbumin
`displays
`the
`best-characterized
`molten
`globule
`state
`and
`is
`the
`best model
`protein
`in protein
`folding
`studies.
`
`a-LACTALBUMIN
`
`(14,200
`protein
`is a small Ca2’-binding
`a-Lactalbumin
`in mammalian
`milk
`and functions
`as a speci-
`Da) present
`ficity
`modifier
`of
`an
`enzyme,
`galactosyltransferase
`to
`(22-24).
`It
`is genetically
`and
`structurally
`homologous
`X-
`c-type
`lysozyme
`(22,
`24-26),
`and
`its high-resolution
`ray crystallographic
`structure
`has
`already
`been
`reported
`(27-30)
`(Fig.
`1). The
`structure
`of a-lactalbumin
`consists
`of
`two subdomains:
`an a-helical
`domain
`and
`a n-sheet
`B
`domain.
`There
`are
`four
`a-helices-A
`[residues
`5-11],
`3jo-
`[23-34],
`C [86-99],
`and D [105-109]-and
`three
`helices
`[12-16,
`101-104,
`and
`115-119]
`a-helical
`
`in the
`
`and reprint
`for correspondence
`tAddress
`of Science,
`of Physics,
`School
`Department
`Japan.
`Hongo, Bunkyo-ku,
`Tokyo
`113,
`CD, circular
`diehroism;
`2Abbreviations:
`NMR; GdnHCl,
`guanidine
`hydrochloride;
`nuclear Overhauser
`effect
`spectroscopy.
`
`to Dr. Kuwajima.
`requests
`University
`of Tokyo,
`
`at:
`7-3-1
`
`2D NMR,
`2D NOESY,
`
`2-dimensional
`2-dimensional
`
`are
`
`a
`
`of
`into
`In
`
`of
`state
`un-
`
`state
`of
`The molten
`a-lactal-
`globule
`ABSTRACI’
`is the best-characterized
`intermediate
`folding
`bumin
`intensively
`studied
`of globular
`and has been
`proteins
`tech-
`by various
`spectroscopic
`and
`physicochemical
`stopped-flow
`CD and
`fluorescence
`niques,
`including
`spectroscopies,
`a hydrogen-exchange
`‘H-
`technique,
`NMR
`spectroscopy,
`disuffide-exchange
`chemistry,
`site-directed
`mutagenesis,
`calorimetric
`tech-
`and
`niques.
`This
`review summarizes
`recent
`studies. Major
`the
`the molten
`globule
`of
`findings
`structure
`about
`1)
`state
`It
`is highly
`heterogeneous,
`are:
`having
`the
`domain
`f3-sheet
`highly
`structured
`a-helical
`with
`and 2) it is not
`domain
`being
`significantly
`unfolded;
`a nonspecific,
`collapsed
`polypeptide
`but
`already
`has
`a native-like
`tertiary
`fold. These
`charac-
`structural
`teristics
`are essential
`to fully understand
`the thermo-
`dynamic
`properties
`of
`the molten
`globule
`state,
`which
`described
`in connection
`with
`a recently
`proposed
`computational
`approach
`to
`predict
`the
`of
`state
`structure
`of
`the molten
`globule
`a protein.
`of
`Mutant
`proteins
`in which
`the
`the molten
`stability
`globule
`state was
`changed
`were
`constructed.
`Studies
`of
`the
`equilibrium
`unfolding
`and
`kinetic
`refolding
`will provide
`further
`insight
`the mutant
`proteins
`the molten
`globule
`state
`as a folding
`intermediate.
`spite
`of
`an
`expectation
`that
`the
`structure
`initial
`an Escherichia
`coli
`recognized
`by
`chaperone,
`is
`GroEL,
`the molten
`globule,
`the
`interaction
`GroEL with a-lactalbumin
`in the molten
`globule
`the
`is much weaker
`than
`interaction
`with more
`folded
`states
`of
`a disulfide-reduced
`ct-lactalbumin,
`form,
`and disulfide
`rearranged
`species.-Kuwajima,
`K.
`The
`molten
`globule
`state
`of
`cx-lactalbu-
`102-109
`(1996)
`min.-FASEBJ.
`
`10,
`
`Key Worth:
`chaperone
`
`folding
`
`intermediate
`
`protein folding
`
`. molecular
`
`studies
`folding
`IN protein
`THE MOST ESSENTIAL
`QUESTION
`can fold
`polypeptide
`of a protein
`is how an unstructured
`period
`conformation
`in a reasonable
`into its unique
`native
`large
`there
`are
`an astronomically
`of
`time
`even
`though
`so-
`conformational
`states
`(1). This
`number
`of possible
`in
`paradox
`has
`directed
`researchers
`called
`Levinthal’s
`inter-
`this
`field
`to try to detect
`characterize
`possible
`and
`states
`mediates
`between
`the
`and
`fully
`unfolded
`native
`(2-11).
`The
`presence
`of a unique
`pathway
`of folding,
`on
`which
`a limited
`number
`of specific
`intermediates
`are well
`populated,
`seems
`to provide
`an answer
`to the
`paradox.
`
`102
`
`Downloaded from
`
`www.fasebj.org
`
` to IP
`
`132.174.254.72
`
`
`
`The FASEB Journal.
`
`0892-6638/96/0010-0102/SOl
` Vol.10,  No.1 , pp:102-109, August, 2017
`
`.50 C FASEB
`
`APOTEX EX1061; Apotex Inc., et al. v. Amgen Inc. et al., IPR2016-01542; Page 1
`
`

`

`a 3io-
`and
`[40-50]
`f’-sheet
`an anti-parallel
`and
`domain;
`and
`The
`structural
`domain.
`in the
`n-sheet
`helix
`[76-82]
`and
`its
`interrela-
`functional
`properties
`of a-lactalbumin
`reviewed
`by
`tionships
`with
`lysozyme
`are
`excellently
`(31). A re-
`McKenzie
`and White
`(24)
`and
`by Kronman
`of protein
`markable
`property
`of a-lactalbumin
`as a model
`globule
`folding
`studies
`is
`the high
`stability
`of its molten
`state
`(4), which
`is observed
`in the
`following
`conditions
`(4): 1) an equilibrium
`unfolding
`intermediate
`at a moder-
`ate
`concentration
`of a strong
`denaturant
`[guanidine
`hy-
`drochloride
`(GdnHC1)
`or urea],
`the
`acid-denatured
`2)
`state,
`and
`3) a partially
`unfolded
`state
`produced
`by re-
`moval
`of
`the bound Ca2+ at neutral
`pH and low salt
`con-
`centration.
`The
`known
`structural
`characteristics
`of
`the
`molten
`globule
`state
`are:
`1) native-like
`secondary
`struc-
`ture,
`2) compact
`structure
`with
`a radius
`only
`10-20%
`3)
`larger
`than
`that
`of
`the
`native molecule,
`and
`the
`ab-
`sence
`of
`the
`specific
`tertiary
`packing
`interactions
`of
`amino
`acid
`side
`chains
`(4). Our group
`studied
`the
`equi-
`librium and
`kinetics
`of
`the unfolding-refolding
`reactions
`among
`the
`three
`states-the
`native,
`the molten
`globule,
`and
`the fully unfolded
`states
`(12, 32, 33)-and
`proposed
`of a-
`a folding model
`based
`on the
`three-state
`unfolding
`tactalbumin
`(34). This model was
`later
`verified,
`using
`a
`kinetic
`CD technique
`(35, 36),
`by direct
`observation
`of a
`transient
`refolding
`intermediate
`that
`is identical
`with the
`molten
`globule
`state.
`These
`studies
`have
`been
`reviewed
`by the
`author
`(4, 15, 16, 37)
`and
`by Sugai
`and
`Ikeguchi
`(38).
`In this
`review,
`only the most
`recent
`progress
`in the
`studies
`of
`the molten
`globule
`state
`of a-lactalbumin
`will
`be described.
`
`HOW NATIVE-LIKE
`GLOBULE?
`
`IS THE MOLTEN
`
`it
`
`secon-
`has native-like
`state
`globule
`the molten
`Although
`far UV CD spectra,
`by the
`dary
`structure
`as measured
`the locations
`of the secondary
`is important
`to ask whether
`the
`polypeptide
`chain
`are
`the
`structure
`segments
`along
`state.
`Dobson
`co-workers
`and
`same
`as
`in the
`native
`ex-
`spectroscopy
`and
`a hydrogen
`(39-41)
`used
`‘H-NMR
`the
`investigating
`the
`structure
`of
`change
`technique
`for
`molten
`globule
`of a-lactalbumin,
`finding
`that
`the B
`state
`and C helices
`are
`formed
`and
`have
`protected
`amide
`pro-
`tons. The
`structure
`of the molten
`globule
`is highly
`hetero-
`geneous,
`having
`the
`highly
`structured
`a-helical
`domain
`formed
`by loose
`hydrophobic
`interactions
`whereas
`the
`-
`sheet
`domain
`is significantly
`more
`unfolded.
`This
`picture
`of the molten
`globule
`state,
`organization
`of the native
`sec-
`ondary
`structure
`segments
`in a part of a protein molecule
`by loose
`tertiary
`contacts,
`is also revealed
`in other
`globu-
`lar
`proteins
`by the
`hydrogen-exchange
`technique
`com-
`bined with
`‘H-NMR
`spectroscopy
`(6,
`17, 19, 42-46).
`It
`is, however,
`important
`to note
`that
`the hydrogen
`exchange
`technique
`can detect
`only native
`secondary
`structure.
`The
`amide
`protons
`protected
`in the
`nonnative
`secondary
`structure,
`if present,
`will
`he
`exchanged
`out during
`
`the
`
`REVIEW
`
`1. Schematic
`representation
`Figure
`generated
`from the structure
`bumin
`1ALC of
`coordinates
`the Brookhaven
`domain
`(residues
`1-37
`and
`85-123)
`domain
`(residues
`38-84)
`in red.
`disulfide
`bonds
`of the protein.
`
`a-lactal-
`of baboon
`of the structure
`proposed
`by Acharya
`et al. (27), using
`Data Bank. The a-helical
`Protein
`is shown
`in blue
`and
`the
`8-sheet
`Four
`yellow sticks
`indicate
`the
`four
`
`the
`
`of
`Formation
`state.
`in the native
`done
`NMR measurement
`has
`refolding
`of
`an early
`stage
`at
`nonnative
`a-helices
`been
`suggested
`in f-lactoglobulin
`by equilibrium
`and ki-
`netic CD studies
`(16, 47).
`2-dimensional
`reported
`Alexandrescu
`et al. have
`(2D NOESY)
`spectroscopy
`nuclear
`Overhauser
`effect
`globule
`state
`(40).
`spectra
`of a-lactalbumin
`in the molten
`of Tyr’#{176}3,Trp’#{176}4,
`They
`have
`shown
`that
`the
`side
`chains
`in the molten
`glob-
`and His’#{176}7form a hydrophobic
`cluster
`region may differ
`ule
`state,
`but
`that
`the
`structure
`in this
`from the
`corresponding
`region
`in the fully
`native
`protein.
`Hydrogen
`exchange
`tryptophan
`indole
`protons,
`photo-
`of
`chemically
`induced
`dynamic
`nuclear
`polarization,
`and
`paramagnetic
`perturbation
`of
`the NMR spectra
`have
`also
`shown
`that Trp26 and Trpt#{176}4,buried
`in the
`native
`state,
`are
`buried
`in the molten
`globule
`(41,
`48)
`so that
`some
`form of a hydrophobic
`core
`in the a-helical
`domain
`of the
`native
`structure
`may persist
`in the molten
`globule
`state.
`Hamada
`et al.
`(49) have
`reported
`that
`the cleavage
`of the
`peptide
`bond
`between Asp
`and Pro67 located
`at
`the edge
`of the f3-sheet
`domain makes
`the
`a-lactalbumin
`molecule
`characteristic
`of
`the molten
`globule
`with
`respect
`to CD
`and
`fluorescence
`spectra
`and
`1-anilino-naphthalene-8-
`shown
`sulfonate
`(ANS)
`binding.
`Nolting
`et at.
`(50)
`have
`at a
`the presence
`of significant
`conformational
`relaxation
`time
`scale
`faster
`than 500 ns in the acidic molten
`globule
`state
`by ultrasonic
`velocimetry.
`the mol-
`to the structure
`Another
`important
`issue
`as
`polypep-
`ten globule
`state
`is:
`Is it a nonspecific
`collapsed
`tide
`or does
`it already
`have
`a native-like
`tertiary
`fold?
`Ikeguchi
`et at.
`(51)
`found
`that
`selective
`reduction
`of
`the
`6-120
`disulfide
`bond
`of bovine
`a-lactalbumin
`reduces
`of
`the
`stability
`the molten
`globule
`state
`by 0.8-1.2
`kcalJmol,
`and have
`suggested
`that
`some
`ordered
`structure
`is present within
`the loop moiety
`formed
`by this disullide.
`
`of
`
`MOLTEN GLOBULE OF a-LACTALBUMIN
`Downloaded from
`
`www.fasebj.org
`
` to IP
`
`132.174.254.72
`
`
`
`The FASEB Journal.
`
` Vol.10,  No.1 , pp:102-109, August, 2017
`
`103
`
`APOTEX EX1061; Apotex Inc., et al. v. Amgen Inc. et al., IPR2016-01542; Page 2
`
`

`

`REVIEW
`
`of
`
`three-
`the
`studied
`have
`(52-54)
`and Ewbank
`Creighton
`that
`and found
`species
`of human
`a-lactalbumin
`disulfIde
`on Cys6 and
`the
`three-disulfide
`form with
`free
`thiols
`Cys12#{176}can
`adopt
`the molten
`globule
`conformation
`and
`then
`spontaneously
`rearrange
`its
`three
`disulfide
`bonds
`to
`many
`isomers
`that maintain
`similar
`conformations
`with
`respect
`to spectroscopic
`and
`hydrodynamic
`properties.
`They
`concluded
`that
`the molten
`globule
`state
`of a-lactal-
`bumin
`does
`not maintain
`the
`native-like
`topology
`of
`the
`polypeptide
`backbone
`but
`is more
`like
`a collapsed
`form
`of an unfolded
`protein.
`co-workers
`by Kim and
`papers
`A series
`recent
`their
`con-
`issue,
`although
`this
`(55-58)
`also
`illuminates
`drawn
`by
`contradictory
`to that
`clusion
`is
`apparently
`a single-
`(54). They
`constructed
`Creighton
`and Ewbank
`chain,
`recombinant
`model
`of the a-helical
`domain
`of hu-
`man
`a-lactalbumin,
`the
`a-domain,
`which
`consists
`of
`residues
`1-39 and 81-123
`of a-lactalbumin
`connected
`by
`a short
`linker
`of
`three
`glycines
`(55). The
`a-domain
`that
`has
`two native
`disullide
`bonds
`(6-120
`and 28-111)
`shows
`characteristics
`of
`the molten
`globule
`state with respect
`to
`the CD and
`‘H-NMR
`spectra
`and
`the
`diffuse
`thermal
`transition,
`showing
`that
`this mini-”protein”
`cannot
`form a
`rigid
`tertiary
`structure
`under
`the
`same
`condition
`that
`the
`intact
`protein
`forms
`the
`native
`structure.
`The
`disulfide-
`exchange
`reaction
`of a-domain
`in a redox
`buffer
`solution,
`however,
`produced
`predominantly
`the
`native
`disulfide
`bonds,
`although
`the other
`two nonnative
`disulfides
`have
`much
`higher
`probabilities
`if they are formed
`in a random-
`pairing model
`of a fully unfolded
`polypeptide.
`The
`results
`clearly
`indicate
`that
`a-domain
`has
`a native-like
`tertiary
`of
`fold
`in spite
`its molten
`globule
`characteristics.
`They
`[a-
`have
`also studied
`two kinds
`of the full-length
`species
`LA(a)
`and
`a-LA()]
`(56).
`a-LA(a)
`contains
`the
`same
`two disullide
`bonds
`in the
`a-domain,
`with
`the
`cyste-
`as
`ines
`for
`the
`other
`two disulfides
`replaced
`by alanines,
`whereas
`a-LA(f)
`contains
`the
`61-77
`disulfide
`in the 3-
`sheet
`domain
`and
`the
`interdomain
`73-91
`disulfide
`bond,
`with
`the
`cysteines
`in the
`a-helical
`domain
`replaced
`by
`alanines.
`a-LA(a)
`shows
`the
`same
`equilibrium
`prefer-
`ences
`of formation
`of the native
`disulfides
`as observed
`in
`the
`a-domain,
`but
`a-LA()
`does
`not
`show a strong
`pref-
`erence
`for
`the native
`disutfide
`fQrmation.
`Thus,
`the mol-
`ten
`globule
`state
`of a-lactalbumin
`has
`a heterogeneous
`structure
`in which
`the a-helical
`domain
`resembles
`an ex-
`panded
`native-like
`protein
`the
`n-sheet
`domain
`is
`and
`largely
`unstructured.
`These
`are consistent
`with the
`results
`hydrogen
`exchange
`results
`of Dobson
`and co-workers
`(41)
`and
`also with
`the
`study
`the
`three-disulfide
`species
`by
`of
`Ikeguchi
`et al.
`(51). The
`apparent
`contradiction
`with the
`results
`of Creighton
`and Ewbank
`(54) may be interpreted
`in terms
`of differences
`in the
`number
`of possible
`disul-
`fide
`pairings
`between
`the
`proteins
`used
`by the
`two
`groups.
`Intact
`a-lactalbumin,
`used
`by Creighton’s
`group,
`contains
`all eight
`cysteines,
`so that
`the preference
`for
`the
`native
`disulfides
`in the
`a-helical
`domain
`may
`be ob-
`scured
`by much more
`random
`distribution
`of disulfide
`pairings
`possible
`in the intact
`protein.
`
`struc-
`the
`reported
`recently
`have
`58)
`(57,
`et al.
`Peng
`specific
`di-
`regions
`surrounding
`local
`of
`tural
`specificity
`state.
`They
`the molten
`globule
`in
`sulfide
`bonds
`determined
`the effective
`concentrations
`for disulfide
`bond
`formation
`for
`the
`two native
`disullide
`bonds
`(6-120
`and
`28-111)
`in the
`a-helical
`domain
`and
`four
`nonnative
`di-
`sulfides
`(6-28,
`6-111,
`28-120,
`and
`111-120)
`by using
`the
`single-disulfide
`mutants
`of full-length
`a-lactalbumin.
`The
`effective
`concentration
`for
`formation
`of the native
`28-111
`disulfide
`bond was more
`than
`10-fold
`higher
`than
`the
`concentration
`for
`formation
`of any other
`native
`or nonna-
`tive disulfide
`bond
`and more
`than
`1000-fold
`higher
`than
`the concentration
`expected
`in the
`random pairing model.
`Thus,
`the
`local
`region
`surrounding
`the 28-111
`disulfide
`bond
`has
`a high
`preference
`for
`adopting
`a native-like
`structure
`in the molten
`globule
`state.
`
`THERMODYNAMIC
`
`PROPERTIES
`
`the
`
`of a-
`state
`globule
`the molten
`of
`property
`A remarkable
`Apparently,
`thermal
`transition.
`its diffuse
`is
`lactalbumin
`in scanning
`heat
`absorption
`peak
`there
`is no cooperative
`is started
`when
`the
`heating
`calorimetric
`measurements
`59,
`60).
`state
`at acid
`pH (35,
`from the molten
`globule
`and the
`that
`the molten
`globule
`This observation
`suggests
`indistin-
`are
`thermodynamically
`thermally
`unfolded
`states
`when we
`is no thermal
`unfolding
`guishable,
`so that
`there
`start
`from the molten
`globule
`state.
`In fact,
`the thermally
`unfolded
`state
`of a-lactalbumin
`shares
`common
`charac-
`teristics
`with
`the molten
`globule
`state-i.e.,
`the
`native-
`like
`secondary
`structure
`and
`the
`compactness
`of
`the
`molecule-especially
`when
`the
`thermal
`unfolding
`is real-
`ized
`at a relatively
`low temperature
`by destabilizing
`the
`native
`state
`(35, 60, 61).
`coopera-
`of
`absence
`the
`of
`Physical
`interpretation
`state
`has,
`globule
`the molten
`of
`tive
`thermal
`transition
`By using multidimensional
`however,
`been
`controversial.
`surface
`obtained
`from scan-
`analysis
`of
`the heat
`capacity
`of a-lactalbumin
`at dif-
`ning
`calorimetric
`measurements
`Xie
`et al.
`have
`ferent
`concentrations
`of GdnHCl,
`(62)
`suggested
`that
`the
`intrinsic
`enthalpy
`change
`from the
`molten
`globule
`to the
`thermally
`unfolded
`state must
`be
`much
`higher
`(24 kcal/mol
`
`at 25#{176}C)than
`that observed
`ex-
`perimentally
`in the
`presence
`of GdnHCl
`or at an acid
`the
`PH;
`it
`is even
`higher
`than
`enthalpy
`change
`from the
`native
`to the molten
`globule
`state
`(only
`8 kcal/mol
`by
`their
`estimate
`at 25#{176}C).This
`conclusion
`has,
`however,
`been
`disputed
`by Yutani
`et al.
`(63), who have
`examined
`the
`heat
`capacity
`function
`of apo-a-lactalbumin
`in the
`molten
`globule
`state
`at neutral
`pH without
`GdnHC1
`by
`differential
`scanning
`microcalorimetry
`and
`have
`shown
`the absence
`of the thermal
`transition.
`Although
`Xie et al.
`(64),
`opposing
`the
`conclusion
`of Yutani
`et al.
`(63),
`later
`attributed
`the
`absence
`of
`the
`thermal
`transition
`to ionic
`strength
`dependence
`the
`thermal
`transition
`tempera-
`of
`ture,
`their
`argument
`is misleading
`because
`thermal
`the
`transition
`observed
`at
`a high
`ionic
`strength
`is not
`the
`
`104 Vol. 10 January
`
`1996
`Downloaded from
`
`www.fasebj.org
`
` to IP
`
`The FASEBJournal
`
`The FASEB Journal.
`132.174.254.72
`
` Vol.10,  No.1 , pp:102-109, August, 2017
`
`KUWAJIMA
`
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`
`

`

`REVIEW
`
`the
`rather
`but
`state
`globule
`from the molten
`transition
`(65).
`state
`from the native
`to the molten
`globule
`transition
`An extensive
`study
`of the
`energetics
`of the a-lactalbu-
`mm states
`has
`recently
`been
`reported
`by Griko
`et al.
`(66). At pH values
`close
`to neutral,
`the thermal
`unfolding
`from the
`native
`state
`occurs
`at high
`temperature
`and
`yields
`a fully
`unfolded
`polypeptide
`with
`no measurable
`population
`of partly
`folded
`intermediates.
`At
`lower
`pH
`at
`values,
`however,
`the
`unfolding
`occurs
`lower
`tempera-
`tures,
`and a progressively
`higher
`population
`of the molten
`globule-like
`intermediates
`was observed.
`Because
`apo-a-
`lactalbumin
`unfolds
`at a low temperature
`even
`at a neu-
`tral pH,
`their
`observation
`of
`the population
`of the molten
`globule
`after
`the
`thermal
`unfolding
`is consistent
`with
`the
`results
`of Yutani
`et al.
`(63). Griko
`et al.
`(66) have
`sug-
`gested
`that
`the molten
`globule
`state
`has a higher
`enthalpy
`than
`the
`unfolded
`state
`below 45#{176}C,so that when
`the
`thermal
`unfolding
`occurs
`below 45#{176}Cthe
`thermally
`un-
`folded
`state
`corresponds
`to the molten
`globule
`state,
`and
`once
`the molten
`globule
`is
`formed,
`the
`structure
`of
`the
`protein
`gradually
`unfolds
`upon
`heating
`and shows
`a grad-
`ual
`increase
`in heat
`capacity.
`by a
`induced
`state
`globule
`The unfolding
`of the molten
`known
`to show
`is, however,
`denaturant,
`urea
`or GdnHC1,
`a cooperative
`transition,
`and Ptitsyn
`and Uversky
`(67)
`re-
`cently
`demonstrated
`the
`transition
`from the molten
`that
`globule
`to the
`fully
`unfolded
`state
`is a two-state
`transi-
`tion.
`The
`thermal
`unfolding
`of a-lactalbumin
`has
`also
`been
`studied
`by fluorescence
`spectroscopy
`and binding
`of
`a hydrophobic
`fluorophor
`(68-72).
`Shimizu
`et al.
`(73)
`have
`studied
`the
`urea-induced
`unfolding
`of
`the molten
`globule
`state
`of a-lactalbumin
`by titration
`of aromatic
`resonances
`in the
`‘H-NMR
`spectra
`and demonstrated
`that
`the
`unfolding
`of
`the molten
`globule
`is not
`a cooperative
`two-state
`process.
`partly
`investigated
`recently
`have
`(74)
`Xie
`and
`Freire
`which
`are
`consistent
`of a-lactalbumin,
`folded
`structures
`data,
`by an inter-
`with
`the
`experimental
`thermodynamic
`generated
`many
`esting
`computational
`approach.
`They
`partly
`folded
`structures
`from the
`known X-ray
`crystal-
`lographic
`structure
`of a-lactalbumin,
`using
`a combinato-
`rial unfolding
`algorithm.
`The
`thermodynamic
`properties
`the partly
`folded
`states
`have
`been
`predicted
`by calculat-
`ing the
`accessible
`surface
`area
`and
`by using
`an empiri-
`cally
`derived
`structural
`parameterization.
`Partly
`folded
`states were
`chosen
`that
`satisfy
`the thermodynamic
`proper-
`the molten
`globule
`state.
`The
`criteria
`used
`to
`ties
`of
`choose
`these
`states
`are a lower heat
`capacity
`and a higher
`enthalpy
`at
`low temperatures
`than
`those
`in the
`fully
`un-
`folded
`state
`(66,
`74).
`Several
`partly
`folded
`states were
`chosen. All of them have
`the A, B, and C helices
`folded,
`whereas
`the p-sheet
`and the D helix were unfolded.
`Their
`results
`are
`thus
`consistent
`with
`the NMR and
`hydrogen
`exchange
`studies
`by Dobso&s
`group
`(41), who
`have
`shown
`the B and C helices
`to be
`formed
`in the molten
`globule
`state. Their
`results
`are,
`however,
`not
`fully consis-
`tent with the
`results
`of Peng
`et al.
`(58), who have
`shown
`that
`the
`region
`surrounding
`the
`28-111
`disulfide
`bond,
`
`of
`
`constructed
`and
`bovine
`residues
`
`in the molten
`is formed
`the D helix,
`which may include
`Xie
`and
`Freire
`globule
`state.
`In all
`their
`calculations,
`(74)
`have
`assumed
`that
`the
`folded
`regions
`in the partly
`folded
`states
`preserve
`the
`conformation
`that
`exists
`in the
`native
`structure.
`This
`is clearly
`an oversimplification.
`Quantitative
`agreement
`in the
`thermodynamic
`parameters
`between
`the
`theory
`and
`experiment,
`they
`reported,
`have
`is thus
`rather
`surprising.
`They
`have
`recently
`applied
`their
`computational
`approach
`to investigating
`the molten
`glob-
`ule states
`of other
`globular
`proteins
`(75-77).
`sta-
`the
`Studies
`of the a-lactalbumin
`mutants
`in which
`bility
`of the molten
`globule
`is changed
`by the muta-
`state
`tions will
`be very
`useful
`for
`elucidating
`the molecular
`mechanism
`of stabilization
`of
`the molten
`globule
`state.
`Recently,
`Uchiyama
`et al.
`(78)
`have
`reported
`studies
`of
`such mutants
`of goat
`a-lactalbumin.
`They
`introduced
`a
`Thr
`to Ile mutation
`at
`residues
`29 and
`33,
`and
`a Ala
`to
`Ile or Ala
`to Thr mutation
`at
`residue
`30. The mutation
`sites
`are located
`in the B helix,
`and the mutations
`change
`the
`hydrophobicity
`of
`the
`core
`of
`the
`a-helical
`domain
`formed
`by the B helix. The
`choice
`of these mutation
`sites
`is based
`on the
`fact
`that guinea
`pig a-lactalbumin
`shows
`a-
`the most
`stable molten
`globule
`state
`among
`the
`three
`lactalbumin
`species
`from goat,
`bovine,
`and
`guinea
`pig.
`The
`stabilization
`free
`energies
`of the molten
`globule
`state
`have
`been
`estimated
`to be 3.1,
`1.5,
`and
`0.7
`kcalJmol
`for
`the guinea
`pig,
`bovine,
`and
`goat
`proteins,
`respectively
`all
`(78).
`The
`amino
`acid
`residues
`at
`these
`sites
`are
`threonines
`in the bovine
`protein;
`residue
`30 is replaced
`by an alanine
`in the goat protein, with the other
`two resi-
`dues
`being
`the
`same
`as
`in the
`bovine
`protein,
`and
`the
`three
`residues
`are
`all
`replaced
`by
`hydrophobic
`isoleucines
`in the guinea
`pig protein.
`The
`stability
`of the
`molten
`globule
`state
`of
`the
`five mutants
`(T29I, A3OT,
`A301, T331,
`and A301+T331)
`of goat
`a-lactalbummn
`has
`been
`investigated
`by urea-induced
`unfolding
`transition
`at
`pH 2.0, where
`the molten
`globule
`state
`is stable without
`urea. The molten
`globule
`state
`is stabilized
`by the amino
`acid
`substitutions
`that
`raise
`the
`hydrophobicity
`of
`the
`residues,
`suggesting
`that
`the hydrophobic
`core
`in a globu-
`lar protein
`plays
`an important
`role
`in stabilization
`of the
`molten
`globule
`state. The
`increase
`in stability
`of the mol-
`ten globule
`state
`is much
`smaller
`than
`the difference
`in
`stability
`between
`the
`guinea
`pig
`and
`goat
`proteins
`and
`less
`than
`10% of
`the
`change
`expected
`from the
`transfer
`free
`energy
`of amino
`acids
`from water
`to hydrophobic
`en-
`vironment,
`e.g.,
`the A301+T331
`double
`mutation
`in-
`the
`creases
`stability
`of
`the molten
`globule
`state
`only
`by
`0.5
`kcal/mol
`(78).
`Thus,
`the
`amino
`acid
`substitutions
`other
`than those
`at
`the three
`sites
`just
`referred
`to are also
`responsible
`for
`the
`remarkable
`stability
`of
`the molten
`globule
`state
`of
`the guinea
`pig protein.
`It has
`also
`been
`concluded
`that
`the hydrophobic
`core
`in the molten
`glob-
`ule
`state
`is highly
`hydrated
`and much
`looser
`than
`the
`core of the native molecule
`(78).
`Pardon
`et al.
`(79)
`have
`recently
`esting
`chimera
`of human
`lysozyme
`bumin.
`In this
`chimera,
`amino
`acid
`
`an inter-
`a-lactal-
`76-102
`
`of
`
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`
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`
`105
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`

`

`REVIEW
`
`lysozyme
`represents
`The
`chimera
`capacity
`and
`globule
`state
`al.
`(79)
`have
`sequence
`of
`the
`stability
`helix
`of
`the
`tant
`features
`sity.
`
`which
`72-97,
`by a-lactalbumin
`replaced
`were
`helix C.
`loop and the central
`the Ca2-binding
`acquired
`strong
`Ca2+binding
`protein
`has
`ability
`to form a stable molten
`shows
`the
`under
`relatively
`mild
`conditions.
`Pardon
`et
`suggested
`that
`the
`peculiar
`aspartate-rich
`the Ca2+binding
`site
`is one
`contribution
`of
`the molten
`globule
`state
`and
`that
`a-lactalbumin
`molecule
`itself
`carries
`that
`determine
`the molten
`globule
`
`to
`the C
`impor-
`propen-
`
`THE MOLTEN
`OF MOLECULAR
`
`AS A MODEL
`GLOBULE
`CHAPERONES
`
`TARGET
`
`has
`
`led to the under-
`chaperones
`of molecular
`Discovery
`phenomena
`are
`cell-biological
`that
`various
`standing
`folding
`(80, 81).
`related
`to the problem of protein
`closely
`various
`chaperone
`proteins,
`the best-characterized
`Among
`the
`from a physicochemical
`point
`of view is
`chaperone
`Da
`coli protein
`GroEL
`(14-mer
`of 60,000
`Escherichia
`which
`is a member
`the
`chaperonin
`60 fam-
`subunits),
`of
`ily. From their
`observation
`of
`in vitro folding
`of dihydro-
`folate
`reductase
`and
`rhodanese
`on the
`surface
`of GroEL,
`Hartl
`and
`co-workers
`(82-84)
`have
`suggested
`that
`the
`conformation
`of GroEL-bound
`polypeptides
`corresponds
`to the molten
`globule
`state
`and have
`proposed
`a model
`which E. coli chaperones-DnaK,
`DnaJ,
`and GroEL-act
`successively
`along
`the
`pathway
`of chaperone-mediated
`protein
`folding
`in vivo. Because
`a-lactalbumin
`exhibits
`the
`best-characterized
`molten
`globule
`state,
`protein
`this
`should
`provide
`a pertinent
`model
`to observed
`directly
`the
`interaction
`between GroEL and the molten
`globule
`state.
`Okazaki
`et al.
`(85)
`have
`thus
`investigated
`the
`interac-
`tions
`between
`GruEL
`and
`two kinds
`of nonnative
`a-lac-
`talbumin-the
`molten
`globule
`state
`and
`the
`disulfide
`reduced
`form-by
`molecular
`sieve
`chromatography
`and
`hydrogen
`exchange
`measurements.
`The molten
`globule
`state was produced
`by removal
`of the bound Ca2+ at neu-
`tral
`pH where GroEL
`is in the
`fully
`native
`state. Disul-
`fide-reduced
`a-lactalbumin
`assumes
`a more
`relaxed
`and
`expanded
`structure
`than
`the molten
`globule
`state
`the
`disulfide-intact
`protein
`(85).
`It
`is
`rather
`surprising
`that
`the
`results
`of Okazaki
`et al.
`(85)
`report
`that
`the
`interac-
`tion between
`GroEL and apo-a-lactalbumin
`in the molten
`globule
`state
`cannot
`be observed
`by molecular
`sieve
`chromatography,
`which
`indicates
`that
`the
`interaction,
`any, must
`be weak. Their
`results
`also show that disullhde-
`reduced
`a-lactalbumin
`is strongly
`bound
`to GroEL when
`50 mM KCI
`is present.
`Therefore,
`the protein
`state
`recog-
`nized
`by GroEL may
`be more
`unfolded
`and
`expanded
`than the typical molten
`globule
`state
`of a-lactalbumin.
`same
`Hayer-Hartl
`et at.
`(86)
`reported
`essentially
`the
`GroEL
`experimental
`results
`of
`the
`interactions
`between
`and
`nonnative
`a-lactalbumin
`as
`reported
`by Okazaki
`et
`at.
`(85),
`but
`their
`conclusion
`is apparently
`contradictory.
`Hayer-Hartl
`et al.
`(85) have
`concluded
`that GroEL
`inter-
`
`in
`
`of
`
`if
`
`to
`real-
`
`in
`by proteins
`exposed
`surfaces
`acts with the hydrophobic
`globule
`state.
`or molten
`a flexible
`compact
`intermediate
`is ascribable
`to
`The discrepancy
`between
`the conclusions
`globule”
`used
`by
`different
`meanings
`of
`the
`term “molten
`the
`two groups.
`Hayer-Hartl
`et al.
`(87)
`are
`using
`this
`term in a wider
`context,
`to include
`the
`conformational
`states
`of disullide-reduced
`or disulfide-rearranged
`species
`of a-lactalbumin.
`Such
`extension
`of the term is, however,
`very confusing
`and misleading
`(88). As we have
`seen
`be-
`fore,
`an important
`characteristic
`of
`the molten
`globule
`state
`is
`its native-like
`tertiary
`fold, which
`is difficult
`realize
`in the
`disulfide-reduced
`species
`and
`never
`ized in the disulfide-rearranged
`species.
`the con-
`Robinson
`et at.
`(89) have
`recently
`investigated
`formation
`of a three-disulfide
`derivative
`of cc-lactalbumin
`ex-
`bound
`to GroEL
`by directly monitoring
`its hydrogen
`spec-
`change
`kinetics
`using
`electrospray
`ionization
`mass
`amide
`trometry.
`The
`bound
`protein
`weakly
`protected
`to that
`protons
`from exchange
`to an extent
`closely
`similar
`three-di-
`of
`of an uncomplexed
`molten
`globule
`state
`the
`sulfide
`protein
`with
`native
`disulfide
`bonds,
`apparently
`a
`suggesting
`that
`the protein
`bound
`to GroEL
`resembles
`of a-
`molten
`globule
`state. The
`three-disulfide
`derivative
`lactalbumin
`they
`used,
`however,
`again was
`the disulfide-
`rearranged
`species. Moreover,
`the
`three-disulfide
`protein
`with
`native
`disulfides
`in the molten
`globule
`is not
`state
`strongly
`bound
`by GroEL
`(86),
`so that once
`the molecule
`acquires
`a native-like
`tertiary
`fold with
`native
`disulfide
`bonds,
`it may not be recognized
`strongly
`by GroEL.
`Re-
`cently,
`we have
`studied
`hydrogen
`exchange
`kinetics
`of
`tritium-labeled
`a-lactalbumin
`with
`the
`four
`disulfide
`bonds
`being
`reduced,
`according
`to tritium-exchange
`fully
`ex-
`measurements
`(Okazaki
`et al. unpublished
`data). The
`of
`change
`kinetics measured
`in the absence
`and presence
`al-
`GroEL
`have
`been
`found
`coincident
`with
`each
`other,
`pro-
`though
`there
`is some
`secondary
`structure
`exhibiting
`of
`tected
`amide
`protons
`in this
`partly
`folded
`state
`a-lactalbumin.
`Thus,
`in spite
`of strong
`binding
`of disul-
`fide
`reduced
`a-lactalbumin
`to GroEL,
`the
`secondary
`by
`structure
`segments
`are
`not
`recognized
`stabilized
`and
`the
`GroEL. Therefore,
`what
`is recognized
`by GroEL is not
`al-
`secondary
`structure
`nor
`a native-like
`tertiary
`fold,
`though
`both are typical
`characteristics
`of the molten
`glob-
`ule state.
`
`CONCLUDING
`
`REMARKS
`
`of
`
`studies
`in the
`progress
`recent
`review summarizes
`This
`structural
`The
`of a-lactalbumin.
`the molten
`globule
`state
`of
`this
`protein
`characteristics
`of
`the molten
`globule
`state
`are
`now much more
`clearly
`defined.
`The most
`important
`characteristic
`emerging
`from recent
`studies
`is the native-
`like
`tertiary
`fold,
`the formation
`of which,
`at
`least
`in a part
`of
`the molecule,
`has
`also
`been
`suggested
`in the molten
`globule
`states
`of other
`globular
`proteins
`(6,
`17,
`19,
`42-46)
`and
`is probably
`a common
`characteristic
`of
`the
`molten
`globule
`state
`for globular
`proteins
`similar
`in size
`
`106 Vol. 10 January
`
`1996
`Downloaded from
`
`www.fasebj.org
`
` to IP
`
`The FASEB Journal
`
`The FASEB Journal.
`132.174.254.72
`
` Vol.10,  No.1 , pp:102-109, August, 2017
`
`KUWAJIMA
`
`APOTEX EX1061; Apotex Inc., et al. v. Amgen Inc. et al., IPR2016-01542; Page 5
`
`

`

`It
`
`of
`
`the
`that
`documented
`also well
`is
`a-lactalbumin.
`pro-
`globular
`in natural
`that
`exist
`folds
`number
`of tertiary
`are classi-
`teins
`is not very large
`and most of the proteins
`fled into only around
`1000
`protein
`families
`(90, 91). As it
`is
`likely
`that
`the
`number
`of
`tertiary
`folds
`is
`limited
`by
`physical
`constraints
`of protein
`structure
`(92),
`an impor-
`tant next
`step
`in protein
`folding
`studies
`is to elucidate
`the
`physical
`constraints
`or interactions
`that are necessary
`and
`sufficient
`to select
`specifically
`one of
`these
`tertiary
`folds
`for a particular
`protein.
`In this
`respect,
`studies
`of the mo-
`lecular mechanism
`of stabilization
`of
`the molten
`globule
`state must be considered
`important.
`the molten
`formation
`It
`is also
`remarkable
`that
`the
`occurs
`very
`globule
`state
`from the
`fully
`unfolded
`state
`rapidly,
`within
`10 ms after
`the refolding
`of a-lactalbumin
`begins.
`Recently,
`we have
`reexamined
`the refolding
`reac-
`tions
`of a-lactalbumin
`from the GdnHCl-induced,
`fully
`unfolded
`state
`by stopped-flow
`CD spectroscopy
`(Arai
`and K. Kuwajima,
`unpublished
`data). An early
`folding
`in-
`termediate
`formed within
`the dead
`time
`(-10
`ms) of the
`stopped-flow
`measurements
`has
`the same
`spectral
`proper-
`ties
`and
`the
`same
`stability
`against
`GdnHC1-induced
`un-
`folding
`as
`the
`equilibrium
`molten
`globule
`state.
`Very
`rapid
`refolding
`within
`an interval
`of a few millisecond
`has
`recently
`been
`observed
`for several
`small
`globular
`proteins
`with approximately
`60 amino
`acid
`residues
`(93-98),
`and
`it has been
`proposed
`that
`the direct
`folding
`to the native
`state
`is a much more
`rapid
`process
`than
`previously
`was
`of a-
`expected
`(98,
`99). Because
`the
`a-helical
`domain
`lactalbumin
`is similar
`in size
`to these
`small
`globular
`pro-
`teins,
`the rapidity
`of formation
`of the molten
`globule
`state
`might
`be analogous
`to the rapidity
`of folding
`of these
`pro-
`teins.
`if
`this
`is the
`case,
`the
`slow folding
`of a-lactalbu-
`mm may be rate-limited
`by rearrangement
`of the
`already
`partly
`organized
`a-helical
`domain
`and
`the more
`unfolded
`n-sheet
`domain.
`the
`and
`(66)
`et at.
`by Griko
`study
`The
`experimental
`seem to
`(74)
`Freire
`by Xie
`and
`theoretical
`calculation
`thermodynamic
`of
`the
`interpretation
`provide
`a reasonable
`the
`Studies
`of
`state.
`characteristics
`of
`the molten
`globule
`the molten
`mutant
`proteins
`that
`stabilize
`or destabilize
`insight
`into the
`globule
`state will provide
`a more
`concrete
`molecular
`interpretation
`of
`the
`thermodynamic
`properties
`and their
`relationship
`to the structure
`i