16 research outputs found
Synthesis of macromolecular systems via lipase catalyzed biocatalytic reactions: applications and future perspectives
Enzymes, being remarkable catalysts, are capable of accepting a wide range of complex molecules as substrates and catalyze a variety of reactions with a high degree of chemo-, stereo- and regioselectivity in most of the reactions. Biocatalysis can be used in both simple and complex chemical transformations without the need for tedious protection and deprotection chemistry that is very common in traditional organic synthesis. This current review highlights the applicability of one class of biocatalysts viz. ââlipasesââ in synthetic transformations, the resolution of pharmaceutically important small molecules including polyphenols, amides, nucleosides and their precursors, the development of macromolecular systems (and their applications as drug/gene carriers), flame retardants, polymeric antioxidants and nanocrystalline solar cells, etc
Secondary Structural Change Can Occur Diffusely and Not Modularly during Protein Folding and Unfolding Reactions
A major
goal of protein folding studies is to understand the structural
basis of the coupling between stabilizing interactions, which leads
to cooperative conformational change. The goal is challenging because
of the difficulty in simultaneously measuring global cooperativity
by determining population distributions of the conformations present,
and the structures of these conformations. Here, hydrogen exchange
(HX) into the small protein monellin was carried out under conditions
where structure-opening is rate limiting for most backbone amide sites.
Detection by mass spectrometry allowed characterization of not only
segment-specific structure-opening rates but also the cooperativity
of unfolding of the different secondary structural segments of the
protein. The segment-specific pattern of HX reveals that the backbone
hydrogen-bonding network disassembles in a structurally diffuse, asynchronous
manner. A comparison of the site-specific transient opening rates
of secondary and tertiary structure in the protein provides a structural
rationale for the observation that unfolding is hierarchical and describable
by exponential kinetics, despite being diffuse. Since unfolding was
studied in native conditions, the sequence of events during folding
in the same conditions will be the reverse of the sequence of events
observed during unfolding. Hence, the formation of secondary structural
units during folding would also occur in a non-cooperative, diffuse,
and asynchronous manner
Tuning Cooperativity on the Free Energy Landscape of Protein Folding
Understanding
the origin of the cooperativity seemingly inherent
in a folding or unfolding reaction has been a major challenge. In
particular, the relationship between folding cooperativity and stability
is poorly understood. In this study, native state hydrogen exchange
in conjunction with mass spectrometry has been used to explore the
free energy landscape accessible to the small protein monellin, when
the stability of the protein is varied. Mass distributions obtained
in the EX1 limit of exchange have allowed a direct distinction between
correlated (cooperative) and uncorrelated (noncooperative) structure-opening
processes. Under conditions where the native protein is maximally
stable, a continuum of partially unfolded states is gradually sampled
before the globally unfolded state is transiently sampled. Under conditions
that stabilize the unfolded state of the protein, the slowest structure-opening
reactions leading to complete unfolding become cooperative. The present
study provides experimental evidence for a gradual uphill unfolding
transition on a very slow time scale, in the presence of a large free
energy difference between the native and unfolded states. The results
suggest that the cooperativity that manifests itself in protein folding
and unfolding reactions carried out in the presence of denaturant
might merely be a consequence of the effect of the denaturant on the
unfolded state and transition state stabilities
High-Energy Intermediates in Protein Unfolding Characterized by Thiol Labeling under Nativelike Conditions
A protein unfolding reaction usually
appears to be so dominated
by a large free energy barrier that identifying and characterizing
high-energy intermediates and, hence, dissecting the unfolding reaction
into multiple structural transitions have proven to be a challenge.
In particular, it has been difficult to identify any detected high-energy
intermediate with the dry (DMG) and wet (WMG) molten globules that
have been implicated in the unfolding reactions of at least some proteins.
In this study, a native-state thiol labeling methodology was used
to identify high-energy intermediates, as well as to delineate the
barriers to the disruption of side chain packing interactions and
to site-specific solvent exposure in different regions of the small
protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing
variants of MNEI have identified three high-energy intermediates,
populated to very low extents under nativelike conditions. A significant
dispersion in the opening rates of the cysteine side chains has allowed
multiple steps, leading to the loss of side chain packing, to be resolved
temporally. A detailed structural analysis of the positions of the
four cysteine residue positions, which are buried to different depths
within the protein, has suggested a direct correlation with the structure
of a DMG, detected in previous studies. It is observed that side chain
packing within the core of the protein is maintained, while that at
the surface is disrupted, in the DMG. The core of the protein becomes
solvent-exposed only in a WMG populated after the rate-limiting step
of unfolding at high denaturant concentrations
Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry
Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other
Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding
To
characterize experimentally the ruggedness of the free energy
landscape of protein folding is challenging, because the distributed
small free energy barriers are usually dominated by one, or a few,
large activation free energy barriers. This study delineates changes
in the roughness of the free energy landscape by making use of the
observation that a decrease in ruggedness is accompanied invariably
by an increase in folding cooperativity. Hydrogen exchange (HX) coupled
to mass spectrometry was used to detect transient sampling of local
energy minima and the global unfolded state on the free energy landscape
of the small protein single-chain monellin. Under native conditions,
local noncooperative openings result in interconversions between Boltzmann-distributed
intermediate states, populated on an extremely rugged âuphillâ
energy landscape. The cooperativity of these interconversions was
increased by selectively destabilizing the native state via mutations,
and further by the addition of a chemical denaturant. The perturbation
of stability alone resulted in seven backbone amide sites exchanging
cooperatively. The size of the cooperatively exchanging and/or unfolding
unit did not depend on the extent of protein destabilization. Only
upon the addition of a denaturant to a destabilized mutant variant
did seven additional backbone amide sites exchange cooperatively.
Segmentwise analysis of the HX kinetics of the mutant variants further
confirmed that the observed increase in cooperativity was due to the
smoothing of the ruggedness of the free energy landscape of folding
of the protein by the chemical denaturant
Synthesis, characterization and in vitro anti-invasive activity screening of polyphenolic and heterocyclic compounds
Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 muM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 muM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells. (C) 2003 Elsevier Science Ltd. All rights reserved