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We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN-S38C-CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments.

We report a general method for light-assisted control of interactions of PDZ domain binding motifs with their cognate domains by the incorporation of a photolabile caging group onto the essential C-terminal carboxylate binding determinant of the motif. The strategy was implemented and validated for both simple monovalent and biomimetic divalent ligands, which have recently been established as powerful tools for acute perturbation of native PDZ domain-dependent interactions in live cells.

Photolabile caging groups, including the 1-(2-nitrophenyl)ethyl (NPE) group, have been applied to probe many biological processes, including protein phosphorylation. Although studies with NPE-caged phosphoamino acids have provided valuable information, these investigations have been limited to the use of only one caged species in a single experiment. To expand the scope of these tools, we have developed an approach for sequentially uncaging two different phosphopeptides in one system, enabling interrogation of multiple phosphorylation events. We present the synthesis of [7-(diethylamino)coumarin-4-yl]methyl (DEACM)-caged phosphorylated serine, threonine, and tyrosine building blocks for Fmoc-based solid-phase peptide synthesis to allow convenient incorporation of these residues into peptides and proteins. Exposure of DEACM- and NPE-caged phosphopeptides to 420 nm light selectively releases the DEACM group without affecting the NPE-caged peptide. This then enables a subsequent irradiation event at 365 nm to remove the NPE group and liberate a second phosphopeptide. We demonstrate the versatility of this general sequential uncaging approach by applying it to control Wip1 phosphatase with two wavelengths of light.

A systematic and general approach for identifying efficient probes for class I PDZ domains based on environment-sensitive chromophores is presented. A series of peptides derived from the C-terminal sequence of Stargazin was first used with PDZ domains of PSD-95 and Shank3 to identify the optimal position and linker length for the 4-DMAP chromophore. The results were applied to well-characterized ligand sequences for each set of domains to generate high affinity probes that retain their native sequence specificity and yield remarkable fluorescence increases upon binding. These probes constitute efficient tools to study the dynamics and regulatory mechanisms of PDZ domain-mediated interactions.

A double-lanthanide-binding tag (dLBT), a small peptide sequence engineered to bind two lanthanide ions (e.g., Tb3+) with high affinity, was used to solve the phase problem for the structure determination of ubiquitin by the single-wavelength anomalous diffraction (SAD) method. Since the dLBT is comprised exclusively of encoded amino acids, the necessity for the incorporation of unnatural amino acids or chemical modification of the protein as a prerequisite for X-ray structure determination is eliminated. A construct encoding the dLBT as an N-terminal fusion with ubiquitin provides for facile expression and purification using standard methods. Phasing of the single-wavelength X-ray data (at 2.6 A resolution) using only the anomalous signal from the two tightly bound Tb3+ ions in the dLBT led to clear electron-density maps. Nearly 75% of the ubiquitin structure was built using automated model-building software without user intervention. It is anticipated that this technique will be broadly applicable, complementing existing macromolecular phasing methodologies. The dLBT should be particularly useful in cases where protein derivatization with heavy atoms proves to be problematic or synchrotron facilities are unavailable.

Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly and selectively complex lanthanide ions and can sensitize terbium (Tb3+) luminescence. On the basis of these properties, it was predicted that increasing the number of bound lanthanides would improve the capabilities of these tags. Therefore, using a structurally well-characterized single-LBT sequence as a starting point, a "double-LBT" (dLBT), which concatenates two lanthanide-binding motifs, was designed. Herein we report the generation of dLBT peptides and luminescence and NMR studies on a dLBT-tagged ubiquitin fusion protein. These lanthanide-bound constructs are shown to be improved luminescent tags with avid lanthanide binding and up to 3-fold greater luminescence intensity. NMR experiments were conducted on the ubiquitin construct, wherein bound paramagnetic lanthanides were used as alignment-inducing agents to gain residual dipolar couplings, which are valuable restraints for macromolecular structure determination. Together, these results indicate that dLBTs will be valuable chemical tools for biophysical applications leading to new approaches for studying the structure, function, and dynamics of proteins.

The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to be consistent with the trends observed from their peptidyl counterparts, though less dramatic because of additional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed the selectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward the glycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.

This protocol presents the synthesis and peptide incorporation of environment-sensitive fluorescent amino acids derived from the dimethylamino-phthalimide family. The procedure uses anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol by these authors. In this study, the corresponding fluorescent amino acids can be readily obtained in Fmoc-protected form for convenient use as building blocks in solid phase peptide synthesis (SPPS). The time required to complete the procedure depends on the size and the number of peptides targeted. Alternatively, the chromophores can be incorporated directly after SPPS via on-resin derivatization of peptides, which is an option described in a related protocol by these authors.

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.

Herein, we report a method for studying protein-peptide interactions which exploits the luminescence properties of Tb(III). Lanthanide-binding tags (LBTs) are short peptide sequences comprising 15-20 naturally occurring amino acids that bind Tb(III) with high affinity. These genetically encodable luminescent tags are smaller in size than the Aequorea victoria fluorescent proteins (AFPs) and benefit from the long-lived luminescence lifetime of lanthanides. In this study, luminescence resonance energy transfer (LRET) was used to monitor the interaction between SH2 domains and different phosphopeptides. For the study, the SH2 domains of Src and Crk kinase were each coexpressed with an LBT, and phosphorylated and nonphosphorylated peptides were chemically synthesized with organic fluorophores. The LRET between the protein-bound Tb(III) and the peptide-based organic fluorophore was shown to be specific for the recognition of the SH2 domain and the peptide binding partner. This method can detect differences in binding affinity and can be used to measure the dissociation constant for the protein-peptide interaction. In addition, decay experiments can be used to calculate the distance between a site in the bound peptide and the protein using Förster theory. In all of these experiments, the millisecond luminescence lifetime of Tb(III) can be exploited using time-resolved detection to eliminate background fluorescence from organic fluorophores.

The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.

Reversible lipid attachment was investigated as a means to deliver small peptides into cells. Two labile straight chain alkyl motifs were developed: a cysteine dodecane disulfide (Cdd) building block and a tyrosine- or serine-myristate ester. Both moieties are cleaved on cell internalization and are compatible with Fmoc solid phase peptide synthesis. A series of fluorophore-labeled peptides that varied in lipophilic content, net charge, and charge distribution were synthesized. The peptides were screened for cellular uptake efficiency as monitored by fluorescence microscopy. Effective peptide transport is based on a distributed net positive charge introduced as lysine residues at the C and/or N terminus of the peptide and the presence of a hydrophobic domain exhibiting an estimated log P4.0. The incorporation of labile lipid motifs into peptides enhances lipophilic character of the peptides and contributes to cellular uptake with minimal alteration to the native sequence.

The study of short, autonomously folding peptides, or "miniproteins," is important for advancing our understanding of protein stability and folding specificity. Although many examples of synthetic alpha-helical structures are known, relatively few mixed alpha/beta structures have been successfully designed. Only one mixed-secondary structure oligomer, an alpha/beta homotetramer, has been reported thus far. In this report, we use structural analysis and computational design to convert this homotetramer into the smallest known alpha/beta-heterotetramer. Computational screening of many possible sequence/structure combinations led efficiently to the design of short, 21-residue peptides that fold cooperatively and autonomously into a specific complex in solution. A 1.95 A crystal structure reveals how steric complementarity and charge patterning encode heterospecificity. The first- and second-generation heterotetrameric miniproteins described here will be useful as simple models for the analysis of protein-protein interaction specificity and as structural platforms for the further elaboration of folding and function.

The x-ray crystal structure of an oligomeric miniprotein has been determined to a 1.2-A resolution by means of multiwavelength anomalous diffraction phasing with selenomethionine analogs that retain the biophysical characteristics of the native peptide. Peptide 1, comprising alpha and beta secondary structure elements with only 21 aa per monomer, associates as a discrete tetramer. The peptide adopts a previously uncharacterized quaternary structure in which alpha and beta components interact to form a tightly packed and well defined hydrophobic core. The structure provides insight into the origins of the unusual thermal stability of the oligomer. The miniprotein shares many characteristics of larger proteins, including cooperative folding, lack of 1-anilino-8-naphthalene sulfonate binding, and limited deuterium exchange, and possesses a buried surface area typical of native proteins.

A new 4-(N,N-dimethylamino) phthalimide-based environment-sensitive fluorescent building block for solid phase peptide synthesis, has been synthesized and incorporated into peptides. Peptides incorporating this residue show great potential for biological applications in sensing protein/protein interactions.

Asparagine-linked glycosylation, the co-translational covalent attachment of carbohydrates to asparagine side chains, has a major effect on the folding, stability, and function of many proteins. The carbohydrate composition in mature glycoproteins is heterogeneous due to modification of the initial oligosaccharide by glycosidases and glycosyltransferases during the glycoprotein passage through the endoplasmic reticulum and Golgi apparatus. Despite the diversity of carbohydrate structures, the core beta-D-(GlcNAc)(2) remains conserved in all N-linked glycoproteins. Previously, results from our laboratory showed that the molecular composition of the core disaccharide has a critical and unique conformational effect on the peptide backbone. Herein, we employ a synergistic experimental and computational approach to study the effect of the stereochemistry of the carbohydrate--peptide linkage on glycopeptide structure. A glycopeptide derived from a hemagglutinin protein fragment was synthesized, with the carbohydrate attached to the peptide with an alpha-linked stereochemistry. Computational and biophysical analyses reveal that the conformations of the peptide and alpha- and beta-linked glycopeptides are uniquely influenced by the attached saccharide. The value of computational approaches for probing the influence of attached saccharides on polypeptide conformation is highlighted.

Three 1-(2-nitrophenyl)ethyl-caged phospho-amino acids have been synthesized for use in standard N(alpha)-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (SPPS). The most common naturally occurring phospho-amino acids, serine, threonine, and tyrosine, were prepared as protected caged building blocks by modification with a unique phosphitylating reagent. In previous work, caged phospho-peptides were made using an interassembly approach (Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B. Org. Lett. 2002, 4, 2865-2868). However, this technique is limited to creating peptides without oxidation sensitive residues C-terminal to the amino acid to be modified and the methodology involves synthetic manipulations on the solid phase that may limit the utilization of the methodology. Herein we report the facile synthesis of N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-serine 1, N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-threonine 2, and N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-tyrosine 3. These building blocks allow the synthesis of any caged phospho-peptide sequence using standard Fmoc-based SPPS procedures.

Herein, we present a methodology that allows for the temporal control of fibrillization of amyloidogenic peptides. This general approach implements a photolabile linker that connects the amyloidogenic peptide to a fibril-inhibitory unit, in this case, a pentamer of amino acids modified with the solubilizing N,N-dimethylethylenediamine (DMDA) units. Upon photolysis, the linker can be dissociated to afford the intact and native amyloidogenic peptide. This methodology should be of value in a variety of studies where spatial and temporal control of supramolecular association processes is desired.

Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb(3+) ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K(D)) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.

Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.

Oligosaccharyl transferase (OT) is the enzyme responsible for asparagine-linked glycosylation in the lumen of the endoplasmic reticulum, which is a subcellular compartment within eukaryotic cells. Inhibition of this enzyme within a cellular environment would provide a valuable investigative tool for glycobiology. Due to the limitations of peptides, none of the existing peptide-based inhibitors of OT demonstrate activity in cell-based enzyme assays. We report herein the design, synthesis and preliminary biological characterization of a family of peptidomimetics that inhibit OT with Ki values in the nanomolar range. The hexapeptide Bz-Dab-Ala-Thr-Val-Thr-Nph-NH2 (Ki = 69 nM) was used as the prototype for the design of bioavailable inhibitors. Several aminobenzoic acid spacer groups were evaluated as potential isosteres of the Val-Thr dipeptide unit and the peptidomimetic incorporating 3-aminobenzoic acid proved to inhibit OT with similar potency to the parent compound (Ki = 84 nM). Further modifications explored the effects of size, hydrophobicity and conformational rigidity on enzyme affinity. This study yielded a family of potent non-peptidic inhibitors that are viable candidates for the in vivo inhibition of OT.

[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.

Here we report the creation of a predominantly beta-structured mini-protein motif. The design target is based on the naturally occurring toxin hand (TH) motifs that are composed of four disulfide bonds and three loops that form a 'hand'. Analysis and subsequent modification of several generations of mini-proteins produced the final 29-residue mini-protein. The structured motif of this new mini-protein provides insight into the compensatory changes that result in the formation of a tightly packed hydrophobic core in a small, globular beta-structure motif. Additionally, this mini-motif represents a new, distinct surface topology for protein design and a valuable, yet compact, model system for the study of beta-sheet structure in water.

A pyridoxamine coenzyme amino acid chimera (Pam) was incorporated into a designed betabeta alpha motif peptide to explore the ability of a small synthetic peptide scaffold to influence coenzyme mediated transamination. Structural characterization of this peptide by CD and NMR spectroscopy suggested that the pyridoxamine containing residue was accommodated into the sheet region of the motif without gross structural perturbations. To investigate the ability of the peptide architecture to influence the amount and distribution of transamination product in the conversion of pyruvic acid to alanine, a family of 18 related peptides, CBP01-CBP18, was rapidly synthesized and purified in parallel. These peptides were designed to generate different peptide environments for the pyridoxamine functionality within the context of the structured betabeta alpha peptide motif. Studies of peptide-mediated transamination revealed clear trends in stereospecific production of L-alanine as a function of substitutions at positions five and seven of the motif. Furthermore, new trends favoring the other enantiomeric product resulted from the addition of copper(II) ion, a known chelator of the transamination reaction intermediates. In the presence of copper(II) ion the amount of alanine product generated was increased by up to 32-fold relative to a pyridoxamine model compound in the presence of copper(II) ion. These functional results, accompanied by further CD and NMR spectroscopic analysis of CBP14, one of the CBP family of peptides, suggest that small synthetic betabeta alpha motif peptides can be used to influence the functional properties of coenzymes.

Background: Small folded polypeptide motifs represented highly simplified systems for theoretical and experimental studies on protein structure and folding. We have recently reported the design and characterization of a metal-ion-independent 23-residue peptide with a beta beta alpha structure (BBA1), based on the zinc finger domains. To understand better the determinants of structure for this small peptide, we investigated the conformational role of the synthetic residue 3-(1, 10-phenanthrol-2-yl)-L-alanine (Fen) in BBA1., Results: NMR analysis revealed that replacing the Fen residue of peptide BBA1 by either of the natural amino acids tyrosine (BBA2) or tryptophan (BBA3) resulted in conformational flexibility in the sheet and loop regions of the structure. This conformational ambiguity was eliminated in peptides BBA4 and BBA5 by including charged residues on the exterior of the beta hairpin designed to both select against the undesired fold and stabilize the desired structure. The evaluation of two additional peptides (BBA6 and BBA7) provided further insight into the specific involvement of the surface polar residues in the creation of well-defined structure in BBA4 and BBA5. The sequences of BBA5, BBA6 and BBA7 include only one non-standard amino acid (D-proline), which constrains a critical engineered type II' turn., Conclusions: Manipulation of residues on the exterior of small beta beta alpha motifs has led to the design of 23-residue polypeptides that adopt a defined tertiary structure in the absence of synthetic amino acids, increasing the availability and expanding the potential uses of the BBA motif. The importance of negative design concepts to the creation of structured polypeptides is also highlighted.

In this paper we present a redesign strategy for the development of uniquely folded polypeptide motifs of less than 40 residues. These mini proteins are based on natural target domains, including the zinc finger domains (BBA motif)* and the disulfide-rich snake and scorpion toxins (BBB motif). These motifs are designed to act as the molecular framework for the construction of novel functional polypeptides. We will explore the structural determinants of the folded BBA motif, inspired by the zinc finger peptides, in relation to the redesign process.

The peptide cyclo(hex-Amb(1)-Cys(2))-Thr(3)-Val(4)-Thr(5)-Nph(6)-NH2 was previously shown to be a slow, tight-binding inhibitor (Ki = 37 nM) of the yeast oligosaccharyl transferase (OT) [Hendrickson et al. (1996) J. Am. Chem. Soc. 118, 7636-7637]. This enzyme catalyzes the transfer of a carbohydrate moiety to an asparagine residue in the consensus sequence Asn-Xaa-Thr/Ser. Herein we present a study of the contribution of the residues in positions 1, 3, 4, and 5 to OT binding. Replacement of the threonine (residue 3) by valine or (S)-2-aminobutyric acid dramatically reduced the potency of the inhibitor while, surprisingly, the incorporation of an additional methylene into the side chain of residue 1 [(S)-2,3-diaminobutyric acid changed to ornithine] had very little effect. Variants with acidic, basic, hydrophilic/polar, and hydrophobic side chains in positions 4 and 5 were also evaluated for both yeast and porcine liver OT inhibition. This aspect of the study reveals that basic (lysine) and acidic (glutamic acid) residues are detrimental to the binding, whereas hydrophobic (valine) and polar/hydrophilic (threonine) residues are both well tolerated. The kinetic behavior of substrate analogs [cyclo(hex-Asn(1)-Cys(2))-Thr(3)-Xaa(4)-Yaa(5)-Nph-NH2] corresponding to inhibitors of weak, medium, and strong potency was also examined in order to provide insight into the nature of these inhibitors.

Small proteins or protein domains generally require disulfide bridges or metal sites for their stabilization. Here it is shown that the beta beta alpha architecture of zinc fingers can be reproduced in a 23-residue polypeptide in the absence of metal ions. The sequence was obtained through an iterative design process. A key feature of the final design is the incorporation of a type II' beta turn to aid in beta-hairpin formation. Nuclear magnetic resonance analysis reveals that the alpha helix and beta hairpin are held together by a defined hydrophobic core. The availability of this structural template has implications for the development of functional polypeptides.

We have synthesized peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases alpha-chymotrypsin and porcine pancreatic elastase. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the gamma-OH group of the active site serine to form a stable hemiacetal [Lowe, G., & Nurse, D. (1977) J. Chem. Soc., Chem. Commun., 815; Chen, R., Gorenstein, D.G., Kennedy, W.P., Lowe, G., Nurse, D., & Schultz, R.M. (1979) Biochemistry 18, 921; Shah, D.O., Lai, K., & Gorenstein, D.G. (1984) J. Am. Chem. Soc. 106, 4272]. 19F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF3 and Ac-ambo-Phe-CF3 clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrate or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of Ki concomitant with the change from a dipeptide analogue to a tetrapeptide analogue (Ac-Pro-ambo-Ala-CF3, Ki = 3 X 10(-3) M; Ac-Ala-Ala-Pro-ambo-Ala-CF3, Ki = 0.34 X 10(-6) M) correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues [Bartlett, P.A., & Marlowe, C.K. (1983) Biochemistry 22, 4618; Thompson, R.C. (1973) Biochemistry 12, 47].(ABSTRACT TRUNCATED AT 250 WORDS)