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In this report, the effect of ionic strength on the loading efficiency of three model polypeptide/protein drugs, namely angiotensin II, insulin, and cytochrome c, in pH- and temperature-sensitive terpolymers of poly(NIPAAm-co-butylmethacrylate-co-acrylic acid) (poly(NIPAAm-co-BMA-co-AA)) has been investigated. Loading efficiency of polypeptides in pH-/temperature-sensitive beads composed of poly(NIPAAm-co-BMA-co-AA) terpolymer is predominantly governed by hydrophobic interactions, both nonspecific surface interactions and/or specific interactions (binding pockets) between the protein and the polymer molecules. Thus, loading efficiency increases with ionic strength. However, as ionic strength increases further, polymer deswelling (collapse), which is also controlled by hydrophobic forces, becomes more pronounced, and consequently, a higher fraction of water is squeezed out during bead formation and the loading efficiency starts to decrease.
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PMID:Effect of ionic strength on the loading efficiency of model polypeptide/protein drugs in pH-/temperature-sensitive polymers. 1068 Jun 7

Poly-N-isopropylacrylamide (PNIPAM) is a chemical isomer of poly-leucine, having the polar peptide group in the side-chain rather than in the backbone. It has been demonstrated experimentally that PNIPAM dissolved in aqueous solution undergoes a collapse transition from coil to globule on increasing temperature above the θ-point. By a careful reviewing of existing experimental data, we emphasize that such coil to globule collapse has to be considered an intramolecular first-order transition, analogous to the cold renaturation of small globular proteins. The main theoretical approaches to the coil to globule collapse in homopolymers are discussed briefly, and a critical comparison between the existing models is performed. We point out that, as a general result, the coil to globule collapse is expected to be a first-order transition for rigid and semi-rigid macromolecules. Finally, taking advantage of the analogy between the coil to globule collapse of PNIPAM and the cold renaturation of small globular proteins, we try to clarify some important and intriguing aspects of protein thermodynamics. This leads to the conclusion that the amphiphilic nature of polypeptide chain plays the fundamental role for the existence of two temperature-induced conformational transitions.
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PMID:On the temperature-induced coil to globule transition of poly-N-isopropylacrylamide in dilute aqueous solutions. 1070 90

The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers and random heteropolymers is continuous and multi-phasic. A new rapid-mixing flow technique has been used to resolve the late stages of polypeptide collapse, at time scales >/=45 microseconds. We have used a laser temperature-jump with fluorescence spectroscopy to resolve the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.
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PMID:Two-state expansion and collapse of a polypeptide. 1073 28

We studied the structure of a polypeptide model by means of the Monte Carlo method. The model chain consisting of two kinds of residues (hydrophobic and hydrophilic) was confined on the (310) hybrid lattice. The residues interacted with the long-range contact potential. The short-range potential was also used by introducing the preferences of conformations corresponding to the helical structure. Simulations of the coil-to-globule collapse were done by an annealing process starting from high-temperature structures and then gradual cooling of the system. The parameters describing the behavior of the system were monitored. It has been found that in a case of a helical pattern -HHPPHPP- the collapsed chains consisted of helical fragments. The resulting structures were formed in such way that the polar residues were located in the outer shell of the molecule since the hydrophobic residues filled the inner part of molecule. The results showed that the proper balance between the magnitude of the potentials used in a model is important and its influence on final structures of molecules was discussed.
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PMID:The effect of sequence patterns and local conformational preferences on the structure of collapsed polypeptide. 1086 34

Immunoglobulin heavy chain binding protein (BiP), a member of the Hsp70 chaperone family, and the oxidoreductase protein-disulfide isomerase (PDI) play an important role in the folding and oxidation of proteins in the endoplasmic reticulum. However, it was not clear whether both cooperate in this process. We show here that BiP and PDI act synergistically in the in vitro folding of the denatured and reduced Fab fragment. Several ATP-dependent cycles of binding, release, and rebinding of the unfolded antibody chains by BiP are required for efficient reactivation. Our data suggest that in the absence of BiP unfolded antibody chains collapse rapidly upon refolding, rendering cysteine side chains inaccessible for PDI. BiP binds the unfolded polypeptide chains and keeps them in a conformation in which the cysteine residues are accessible for PDI. These findings support the idea of a network of folding helper proteins in the endoplasmic reticulum, which makes this organelle a dedicated protein-processing compartment.
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PMID:BiP and PDI cooperate in the oxidative folding of antibodies in vitro. 1089 9

This review describes how kinetic experiments using techniques with dramatically improved time resolution have contributed to understanding mechanisms in protein folding. Optical triggering with nanosecond laser pulses has made it possible to study the fastest-folding proteins as well as fundamental processes in folding for the first time. These include formation of alpha-helices, beta-sheets, and contacts between residues distant in sequence, as well as overall collapse of the polypeptide chain. Improvements in the time resolution of mixing experiments and the use of dynamic nuclear magnetic resonance methods have also allowed kinetic studies of proteins that fold too fast (greater than approximately 10(3) s-1) to be observed by conventional methods. Simple statistical mechanical models have been extremely useful in interpreting the experimental results. One of the surprises is that models originally developed for explaining the fast kinetics of secondary structure formation in isolated peptides are also successful in calculating folding rates of single domain proteins from their native three-dimensional structure.
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PMID:Fast kinetics and mechanisms in protein folding. 1094 Feb 52

The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers is continuous and multi-phasic. We have used a laser temperature-jump with fluorescence spectroscopy to measure the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.
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PMID:Two-state expansion and collapse of a polypeptide. 1096 3

Elastin-like polypeptides (ELPs) are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. They are soluble in aqueous solutions below their transition temperature (T1) but hydrophobically collapse and aggregate at temperatures greater than T1. We hypothesized that ELPs conjugated to drugs would enable thermally targeted drug delivery to solid tumors if their T1 were between body temperature and the temperature in a locally heated region. To test this hypothesis, we synthesized a thermally responsive ELP with a T1 of 41 degrees C and a thermally unresponsive control ELP in Escherichia coli using recombinant DNA techniques. In vivo fluorescence videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors (SKOV-3 ovarian carcinoma and D-54MG glioma) implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately 2-fold increase in tumor localization compared to the same polypeptide without hyperthermia. We observed aggregates of the thermally responsive ELP by fluorescence videomicroscopy within the heated tumor microvasculature but not in control experiments, which demonstrates that the phase transition of the thermally responsive ELP carrier can be engineered to occur in vivo at a specified temperature. By exploiting the phase transition-induced aggregation of these polypeptides, this method provides a new way to thermally target polymer-drug conjugates to solid tumors.
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PMID:Targeting a genetically engineered elastin-like polypeptide to solid tumors by local hyperthermia. 1124 64

Intestinal fatty acid binding protein (IFABP) is a member of the lipid binding protein family, members of which have a clam shell type of motif formed by two five-stranded beta-sheets. Understanding the folding mechanism of these proteins has been hindered by the presence of an unresolved burst phase. By initiating the reaction with a sub-millisecond mixer and following its progression by Trp fluorescence, we discovered three distinct phases in the folding reaction of the W6Y mutant of IFABP from which we postulate the following sequence of events. The first phase (k(1) > 10 000 s(-1)) involves collapse of the polypeptide chain around a hydrophobic core. During the second phase (k(2) approximately 1500 s(-1)), beta-strands B-G, mostly located on the top half of the clam shell structure, propagate from this hydrophobic core. It is followed by the final phase (k(3) approximately 5 s(-1)) involving the formation of the last three beta-strands on the bottom half of the clam shell and the establishment of the native hydrogen bonding network throughout the protein molecule.
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PMID:Hierarchical folding of intestinal fatty acid binding protein. 1128 75

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.
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PMID:NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding. 1129 22


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