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L N Lin

Publications and source records attributed to L N Lin.

32 records · Page 2Linked to original sources
PubMed1987

Refolding of ribonuclease in the presence and absence of ammonium sulfate pulses. Comparison between experiments and simulations.

Experiments have been carried out on ribonuclease A in which refolding in high concentrations of guanidine hydrochloride is either preceded or not preceded by a short ammonium sulfate pulse. Application of the pulse causes the rapid formation of the nativelike intermediate, and the effect of this pulse was determined by using three different methods for monitoring the subsequent refolding reaction: direct absorbance, direct fluorescence, and a double-jump fluorescence unfolding assay which is specific for the isomerization of proline-93. The effect of the pulse is quite different depending on the method of detection. With absorbance detection, the pulse causes a large reduction in the refolding amplitude with no change in the kinetics of the decay curve, while with the fluorescence unfolding assay, the pulse causes no change in the refolding amplitude but produces a large acceleration in the decay kinetics. The results with direct fluorescence are intermediate with some reduction seen in the refolding amplitude and some acceleration in the decay kinetics. The results of these experiments are simulated by using the simple model of Lin and Brandts (1984) [Lin, L.-N., & Brandts, J. F. (1984) Biochemistry 23, 5713] in which proline-93 must be in the correct cis configuration before folding to the native or nativelike state can occur. In all cases, the simulations accurately predict the experimental results for all three methods of detection, without any adjustment of parameter values from those published earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗
PubMed1985

Isomer-specific proteolysis of model substrates: influence that the location of the proline residue exerts on cis/trans specificity.

In an effort to further develop the technique of isomer-specific proteolysis, a number of proline-containing substrates were subjected to hydrolysis in the presence of chymotrypsin, trypsin, or prolidase. The objective was to determine whether direct hydrolysis of the cis form of the substrate could occur and, if so, the extent to which it is slower than the hydrolysis of the equivalent trans form. It is shown that for both peptide and amide substrates, which contain proline at the P2 position, the cis form can be hydrolyzed directly by either chymotrypsin or trypsin, in contrast to earlier suggestions in the literature. For similar amide substrates, it was found that chymotrypsin has a lower catalytic efficiency for the cis form, relative to the trans form, by a factor of 20 000 while, for trypsin and its substrate, the cis form was cleaved about 2000 times less efficiently. Results for a trypsin substrate with proline at the P2' position, rather than the P2 position, were quite different however, since there was no indication that the cis form could be directly cleaved even at the highest enzyme concentration. There was also no indication that prolidase could cleave the dipeptide Phe-Pro when the active bond itself is in the cis form. These collective results suggest that the ability of proteases to cleave a substrate with a cis peptide bond depends strongly on the location of the cis bond relative to the active bond that is being cleaved.

Amino Acid Sequence↗
PubMed1984

Involvement of prolines-114 and -117 in the slow refolding phase of ribonuclease A as determined by isomer-specific proteolysis.

Using the method of isomer-specific proteolysis (ISP), the cis-trans nature of the peptide bonds involving prolines-114 and -117 in ribonuclease (RNase) has been investigated. These studies involve the pretreatment of RNase first with either a short pepsin pulse or a short mercaptoethanol pulse to irreversibly unfold the protein and then with a short chymotrypsin pulse to quickly cleave the Tyr115-Val116 bond so that the chain is suitably trimmed for the subsequent stereospecific cleavage either by aminopeptidase P, to investigate proline-117, or by a proline-specific endopeptidase, to investigate proline-114. The most reasonable interpretation of our results suggests that proline-117 is essentially 100% trans in both the native and unfolded states, so it apparently makes no direct contribution to the slow refolding kinetics of RNase. It is also determined that proline-114 is 100% cis in native RNase and ca. 95% cis in reversibly unfolded RNase so only 5% of the unfolded RNase can be rate limited by trans to cis isomerization of proline-114 during refolding. Careful spectroscopic studies of refolding show that the smallest and slowest of the refolding phases, the ct phase, has the proper amplitude (5%), relaxation time (400 s at 10 degrees C), and activation energy (17 kcal) for a phase that is rate limited by the trans to cis isomerization of proline-114. Measurements of the kinetics of binding of cytidine 2'-monophosphate during refolding further show that RNase does not become active until proline-114 has isomerized to the native cis configuration. It is concluded that none of the three prolines thus far examined (i.e., prolines-93, -114, and -117) by the ISP method is involved in the formation of a fully active, nativelike intermediate which has "incorrect" proline isomers. The specific structural process which is responsible for the largest of the three slow refolding phases, the XY phase, is still undetermined. Although ISP results on proline-42 are not yet available, it seems possible that this slow phase may be rate limited by a process other than proline isomerization. In unrelated studies, results from chymotrypsin hydrolyses of several short peptides containing the sequence -X-Y-Pro- show that cleavage of an active X-Y bond is very slow when it is immediately adjacent on the amino side of a proline peptide bond. Thus, chymotrypsin cleavage may not be generally useful as the analytical step in isomer-specific proteolysis.

Amino Acid Sequence↗
PubMed1983

Evidence showing that a proline-specific endopeptidase has an absolute requirement for a trans peptide bond immediately preceding the active bond.

The proline-specific endopeptidase (EC 3.4.21.26) from Flavobacterium meningosepticum is specific for the cleavage of peptide bonds on the C-terminal side of prolyl residues. Such bonds will normally exist in the all-trans configuration. However, the preceding peptide bond in the sequence (i.e., on the N-terminal side of the prolyl residue) will exist as a mixture of cis and trans forms in solution. In this study, the activity of the proline-specific endopeptidase toward the substrates N-Cbz-Gly-Pro-MCA (where MCA = 4-methylcoumarinyl-7-amine) and N-Cbz-Gly-Pro-Leu-Gly has been examined. At a high ratio of enzyme activity/substrate concentration, the hydrolysis pattern for each substrate shows two well-separated kinetic phases. It is concluded that the fast kinetic phase, whose velocity depends on enzyme concentration, results from the direct hydrolysis of the active substrate bond (i.e., either the Pro-MCA or Pro-Leu bond, respectively) in molecules where the preceding Gly-Pro bond is trans. The slow phase, whose velocity is independent of enzyme concentration, is rate-limited by the cis-to-trans isomerization of those substrate molecules which initially have the preceding Gly-Pro bond in the cis configuration. That is, substrate molecules having the cis form of the Gly-Pro bond which precedes the active bond cannot be hydrolyzed directly but must first isomerize to the trans form before cleavage can occur. The amplitude, relaxation time, and activation energy for the slow phase are consistent with this interpretation. Thus, the proline-specific endopeptidase from Flavobacterium has an absolute requirement for a trans peptide bond at the position immediately preceding the active bond.

Amino Acid Sequence↗
PubMed1983

Determination of cis-trans proline isomerization by trypsin proteolysis. Application to a model pentapeptide and to oxidized ribonuclease A.

It is shown, by examination of a model pentapeptide, that trypsin will only cleave substrate bonds in a polypeptide chain when the peptide bond following the active bond is in the trans isomeric state. The cis form must isomerize to trans before it can be cleaved. Taking advantage of this isomeric specificity, the sequence-Lys91-Tyr92-Pro93- is examined in oxidized RNase A. It is shown that the Tyr-Pro bond exists 33% in the cis form at equilibrium and that the cis-to-trans relaxation time for isomerization is 5.0 min at 10 degrees C. The fragment 92-98 has about the same cis content (35%) as does oxidized RNase A but has a much slower relaxation time (11 min). This suggests that overall chain dynamics may exert some effect on the kinetics of isomerization.

Amino Acid Sequence↗
PubMed1983

Isomerization of proline-93 during the unfolding and refolding of ribonuclease A.

Using the method of isomer-specific proteolysis, the isomerization of proline-93 has been monitored directly during the time course of the unfolding and refolding reactions of RNase A. It has been found that proline-93 is 100% cis in the native protein and 70% cis in the reversibly unfolded protein. During the unfolding reaction, the change from 100% to 70% cis occurs as a first-order process with a relaxation time of 140 s in 8.5 M urea, 10 degrees C. For refolding, the change from 70% to 100% cis also occurs as a first-order process, with a relaxation time (10 degrees C) of 90 s in 0.3 M urea, 130 s in 1.0 M urea, and 310 s in 2.0 M urea. Parallel experiments which measured the recovery of enzyme activity during refolding were also conducted. These show that 30% of the activity recovers in a slow phase with a first-order relaxation time (10 degrees C) of 100 s in 0.3 M urea. Because of the excellent agreement of both the amplitude and relaxation time for trans-to-cis isomerization and for activity recovery, it is concluded that the slowest phase in the recovery of enzyme activity is rate limited by the isomerization of proline-93. These results demonstrate that proline-93 must be cis before refolding to the active form can take place, in contrast to previous suggestions, and argue against the existence of a nativelike intermediate form on the refolding pathway which contains proline-93 in the incorrect trans configuration.

Animals↗
PubMed1979

Evidence suggesting that some proteolytic enzymes may cleave only the trans form of the peptide bond.

The rates of hydrolysis of glycy-L-proline and L-phenylalanyl-L-proline, catalyzed by prolidase, have been measured at several temperatures under conditions where a high ratio of prolidase activity to substrate concentration existed. Two well-separated kinetic phases, which can be adequately treated as two first-order reactions, were observed for the hydrolysis. The relative amplitudes of the two phases are nearly independent of temperature, but strongly dependent on the initial state of protonation of the dipeptides. It was found that the amplitude of the slow phase is strictly proportional to the known amount of cis isomer, while the amplitude of the fast phase correlates with the amount of the trans isomer. Furthermore, the relaxation time and activation energy of the slow phase of hydrolysis are in good agreement with the same parameters determined for cis-trans isomerization of the dipeptides, as measured by a pH-jump method for samples not being hydrolyzed. These results lead us to the conclusion that the slow phase seen for hydrolysis is rate limited by cis-trans isomerization of the X-pro peptide bond. Thus, this proline-specific protease appears to have an absolute requirement for the trans form of the peptide bond and appears not to cleave the cis form or to cleave it extremely slowly.

Animals↗