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The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 [micro]L of 1 x phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/ mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases. 10.1021/ac101462c

Single-molecule fluorescence spectroscopy (SMFS) is used to show the existence of fluctuations in the activity of single [alpha]-chymotrypsin enzymes and also to show that, during its deactivation, the enzyme is altered stepwise before it deactivates irreversibly. The analysis of the chymotrypsin dynamics has not only revealed dynamic disorder during the active period but has also shown that the deactivation occurs stepwise, rather than by an all-or-nothing event.

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70-80 loop and the unique $1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by [Zn.sup.2+] and [Cu.sup.2+] at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at [His.sup.99] close to the catalytic triad accounts for the noncompetitive [Zn.sup.2+] inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition. 99 loop | desquamation | metal-binding proteinase | Netherlon syndrome

Ab initio simulations of the folding pathways are currently limited to very small proteins. For larger proteins, some approximations or simplifications in protein models need to be introduced. Protein folding and unfolding are among the basic processes in the cell and are very difficult to characterize in detail by experiment or simulation. Chymotrypsin inhibitor 2 (CI2) and barnase are probably the best characterized experimentally in this respect. For these model systems, initial folding stages were simulated by using CA-CB-side chain (CABS), a reduced-space protein-modeling tool. CABS employs knowledge-based potentials that proved to be very successful in protein structure prediction. With the use of isothermal Monte Carlo (MC) dynamics, initiation sites with a residual structure and weak tertiary interactions were identified. Such structures are essential for the initiation of the folding process through a sequential reduction of the protein conformational space, overcoming the Levinthal paradox in this manner. Furthermore, nucleation sites that initiate a tertiary interactions network were located. The MC simulations correspond perfectly to the results of experimental and theoretical research and bring insights into CI2 folding mechanism: unambiguous sequence of folding events was reported as well as cooperative substructures compatible with those obtained in recent molecular dynamics unfolding studies. The correspondence between the simulation and experiment shows that knowledge-based potentials are not only useful in protein structure predictions but are also capable of reproducing the folding pathways. Thus, the results of this work significantly extend the applicability range of reduced models in the theoretical study of proteins. protein structure prediction | Monte Carlo simulations | protein denatured state | folding nucleus | residual structure

The interaction of water-soluble gold nanoparticles with alpha-chymotrypsin is investigated by activity assay, gel electrophoresis, zeta-potential, circular dichroism, and fluorescence spectroscopy. Results reveal that both electrostatic and hydrophobic interactions between the hydrophobic patches of receptors and the protein contribute to the stability of the complex.

Hybrid density functional quantum mechanical calculations were used to study the strength of the hydrogen bond between His(super 57)N(super delta1) and Asp(super 102)O(super delta1) in chymotrypsin and how it changes along the reaction coordinate. The results confirm the hypothesis that the strengthened hydrogen bond plays an important role in lowering the energy of the transition state and hence, in the catalytic efficiency of the enzyme.

The mechanisms of enzymatic degradation of A[beta] with cross-[beta] structures and the series of steps involved in the digestion of A[beta] microfibrils to nanospheres or nanofilaments by protease XIV or [alpha]-chymotrypsin, respectively are reported. The degradation products which exhibit almost the same secondary structures but different cytotoxicities indicated that relationships between nanoassembled structures and cytotoxicity of A[beta] peptides are more significant than the [beta]-sheet content.

A series of proteasomal active site mutants in yeast cell lysates are used to assess the highly selective proteasomal chymotrypsin-like sensor prepared via a combinatorial library strategy. The activities of chymotrypsin-like sensor are enhanced in the presence of the trypsin-like substrate but reduced in the presence of caspase-like substrate and the sensor was also found to hinder the activity of both caspase- and trypsin-like active sites.

The significance of the serine protease, prostrate-specific antigen (PSA), in the development and progression of prostate cancer is analyzed. PSA is shown to exhibit chymotrypsin-like substrate specificity and hence can be easily used as effective therapeutic or imaging agents.

An NMR method is described that has distinguished viscosity effects from binding and self-association in crowded solutions by using a model system that contains millimeter concentrations of chymotrypsin inhibitor 2 and in bovine serum albumin. The results have highlighted the importance of choosing an inert crowding agent to analyze the effects of macromolecular crowding.

Point mutations have caused members of the serine protease inhibitor (serpin) superfamily to undergo a novel conformational transition, forming ordered polymers that have characterized a group of diseases termed the serpinopathies. Studies have shown that serpins can even play a noninhibitory role in events as diverse as blood pressure regulation, chromatin condensation, tumor progression and hormone transport.

Extensive NMR studies were performed to determine the effects of hydrophobic core mutations in chymotrypsin inhibitor 2 (CI2) on the internal dynamics of both the backbone and methyl-containing side-chains. The observed dynamic similarities of CI2 mutants to each other than to the more rigid wild-type CI2 revealed that optimization of wild-type CI2 to a specific level of rigidity might aid CI2 in its function as a serine protease inhibitors.

The various differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein are studied. The analysis of the flexible side chains is shown to be extremely helpful for the in-cell NMR of ordered proteins.

A simple and efficient photo switching method to modulate binding of [alpha]-chymotrypsin to a surface-attached inhibitor is demonstrated.

A new amphiphilic homopolymer that binds noncovalently to proteins is reported. This polymer not only binds to the target protein chymotrypsin with submicromolar affinity but also stabilizes the native structure of the protein.

A computational QM/MM methodology for the systematic study of various structural and spectroscopic properties of strong hydrogen bonds in enzymes is presented. The theoretical model is applied to the N(super delta1)-H-O(super delta1) hydrogen bond between His57 and Asp102 in the active sites of low-pH alpha-chymotrypsin and alpha-lytic protease.