Impact of early incentive spirometry in an enhanced recovery program after laparoscopic donor nephrectomy.

BACKGROUND: This study aimed to assess the impact of early incentive spirometry on the incidence of chest infection in patients undergoing laparoscopic donor nephrectomy. METHODS: A retrospective review on all consecutive laparoscopic donor nephrectomies (LDN) performed at a single institution from January 2008 to August 2012 was performed. We performed 84 LDN. Seventy patients had epidural analgesia continued for 48 hours postoperatively and 14 had a combination of spinal followed by oral analgesia. Incentive spirometry was introduced from July 2010 and 45 of the 84 donors used the spirometer as taught, both pre- and postoperatively. RESULTS: We performed 84 LDN; 39 patients did not receive incentive spirometers and had postoperative chest physiotherapy started on postoperative day 1. Of the 45 patients given incentive spirometers, 44 started using their spirometers as taught, after recovery once they were settled in the ward, 1 patient started the exercises the following day. In the group who received no spirometer, 5 patients had a chest infection. In the group of patients who started using their spirometers in the early perioperative period (44/45), no patient developed a chest infection. One patient in this group was excluded from the analysis because he started spirometer exercises on postoperative day 1. This patient did develop a chest infection. CONCLUSIONS: Our results suggest that early introduction of incentive spirometry after LDN significantly reduces the incidence of chest infection (P < .05); however, this benefit may be lost if the introduction of spirometry is delayed.

miR-125b promotes cell death by targeting spindle assembly checkpoint gene MAD1 and modulating mitotic progression.

The spindle assembly checkpoint (SAC) is a 'wait-anaphase' mechanism that has evolved in eukaryotic cells in response to the stochastic nature of chromosome-spindle attachments. In the recent past, different aspects of the SAC regulation have been described. However, the role of microRNAs in the SAC is vaguely understood. We report here that Mad1, a core SAC protein, is repressed by human miR-125b. Mad1 serves as an adaptor protein for Mad2 - which functions to inhibit anaphase entry till the chromosomal defects in metaphase are corrected. We show that exogenous expression of miR-125b, through downregulation of Mad1, delays cells at metaphase. As a result of this delay, cells proceed towards apoptotic death, which follows from elevated chromosomal abnormalities upon ectopic expression of miR-125b. Moreover, expressions of Mad1 and miR-125b are inversely correlated in a variety of cancer cell lines, as well as in primary head and neck tumour tissues. We conclude that increased expression of miR-125b inhibits cell proliferation by suppressing Mad1 and activating the SAC transiently. We hypothesize an optimum Mad1 level and thus, a properly scheduled SAC is maintained partly by miR-125b.

Lipopolysaccharide neutralizing peptide-porphyrin conjugates for effective photoinactivation and intracellular imaging of gram-negative bacteria strains.

A simple and specific strategy based on the bioconjugation of a photosensitizer protophophyrin IX (PpIX) with a lipopolysaccharide (LPS) binding antimicrobial peptide YI13WF (YVLWKRKRKFCFI-Amide) has been developed for the effective fluorescent imaging and photodynamic inactivation of Gram-negative bacterial strains. The intracellular fluorescent imaging and photodynamic antimicrobial chemotherapy (PACT) studies supported our hypothesis that the PpIX-YI13WF conjugates could serve as efficient probes to image the bacterial strains and meanwhile indicated the potent activities against Gram-negative bacterial pathogens especially for those with antibiotics resistance when exposed to the white light irradiation. Compared to the monomeric PpIX-YI13WF conjugate, the dimeric conjugate indicated the stronger fluorescent imaging signals and higher photoinactivation toward the Gram-negative bacterial pathogens throughout the whole concentration range. In addition, the photodynamic bacterial inactivation also demonstrated more potent activity than the minimum inhibitory concentration (MIC) values of dimeric PpIX-YI13WF conjugate itself observed for E. coli DH5a (~4 times), S. enterica (~8 times), and other Gram-negative strains including antibiotic-resistant E. coli BL21 (~8 times) and K. pneumoniae (~16 times). Moreover, both fluorescent imaging and photoinactivation measurements also demonstrated that the dimeric PpIX-YI13WF conjugate could selectively recognize bacterial strains over mammalian cells and generate less photo damage to mammalian cells. We believed that the enhanced fluorescence and bacterial inactivation were probably attributed to the higher binding affinity between dimeric photosensitizer peptide conjugate and LPS components on the surface of bacterial strains, which were the results of efficient multivalent interactions.

De novo designed lipopolysaccharide binding peptides: structure based development of antiendotoxic and antimicrobial drugs.

Lipopolysaccharide (LPS), the glycolipid of the outer membrane of Gram-negative bacteria, is critically involved in health and diseases. LPS facilitates the survival of pathogens by imposing a permeability barrier against antibiotics and antimicrobial peptides. LPS, also termed as endotoxin, functions as a potent inducer of innate immunity. Interception of endotoxin in systemic circulation by immune cells e.g. macrophages is essential to mount surveillance against invading microbes. However, a hyper-activated immune response may lead to the overwhelming production of tissue damaging cytokines TNF-α, IL-1, IL-6 and free radicals that may cause multiple organ failures or septic shock syndromes. The sepsis or septic shock is the major cause of mortality; 120,000 deaths/year occur in the United States alone, in the intensive care units. To-date, no therapeutic is available to combat sepsis mediated lethality. Furthermore, bacterial resistance against commonly used antibiotics has been increasing at an alarming rate necessitating a search for antibacterial agents with novel mode of actions. LPS could be a valid drug target for the development of antiendotoxic and antimicrobial compounds. In this article, recent advances in structural basis of LPS recognition by its receptor proteins and mode of actions of antimicrobial peptides defensins and cathelicidins are reviewed. Our research has identified, through de novo design, antimicrobial and endotoxin interacting β-boomerang peptides. Structure-activity correlations (SAR) of these peptides have been discussed, highlighting future design to achieve potent LPS neutralizing molecules.

Intensive follow-up after liver resection for colorectal liver metastases: results of combined serial tumour marker estimations and computed tomography of the chest and abdomen - a prospective study.

The aim of the study was to prospectively evaluate an intensive follow-up programme using serial tumour marker estimations and contrast-enhanced computed tomography (CT) of the chest and abdomen in patients undergoing potentially curative resection of colorectal liver metastases. Seventy-six consecutive patients having undergone potentially curative resections of colorectal liver metastases in a single unit were followed up with a protocol of 3 monthly carcinoembryonic antigen and carbohydrate antigen 19-9 estimations and contrast-enhanced spiral CT of the chest, abdomen and pelvis for the first 2 years following surgery and 6 monthly thereafter. The median period of follow-up was 24 months (range 18-60). Recurrent tumour was classed as early if within 6 months of liver resection. Thirty-seven of the 76 patients (49%) developed recurrence on follow-up. Nineteen recurrences were in the liver alone (51%), 16 liver and extrahepatic (43%) and two extrahepatic alone (6%). Of the 19 patients with isolated liver recurrence, eight developed within 6 months of liver resection none of which were resectable. Of the 11 recurrences after 6 months, five (45%) were resectable. Of the 37 recurrences, CT indicated recurrence despite normal tumour markers in 19 patients. Tumour markers suggested recurrence before imaging in 12 and concurrently with imaging in 6. In the 12 patients who presented with elevated tumour markers before imaging, there was a median lag period of 3 months (range 1-21) in recurrence being detected on further serial imaging. Seventeen patients who developed recurrence had normal tumour markers before initial resection of their liver metastases. Of these 17, 10 (58%) had an elevation of tumour markers associated with recurrence. Over a median follow-up of 2 years following liver resection, the use of CT or tumour markers alone would have failed to demonstrate early recurrence in 12 and 18 patients respectively. A combination of tumour markers and CT detected significantly more (P < 0.05) recurrence than either modality alone. Tumour markers and CT should be used in combination in the follow-up of patients with resected colorectal liver metatases, including patients whose markers are normal at the time of initial liver resection.

Prospective study of contrast-enhanced computed tomography, computed tomography during arterioportography, and magnetic resonance imaging for staging colorectal liver metastases for liver resection.

BACKGROUND: This study compared the value of contrast-enhanced helical computed tomography (CT), CT during arterioportography (CTAP), and contrast-enhanced magnetic resonance imaging (MRI) for staging patients with colorectal liver metastases. METHODS: One hundred and twenty patients with known or suspected colorectal liver metastases were evaluated prospectively. MRI and CTAP were performed within 3 weeks of CT in patients with potentially resectable tumours. Results of imaging were compared with findings at surgery, intraoperative ultrasonography and histological examination. RESULTS: Twenty patients were not considered for liver resection following CT. The remaining 100 patients underwent CT and CTAP, 85 of whom had CT, CTAP and MRI. The sensitivity and specificity were 73.0 and 96.5 per cent for CT, 87.1 and 89.3 per cent for CTAP, and 81.9 and 93.2 per cent for MRI. Positive predictive values were 89.7, 87.5 and 87.5 per cent respectively. Receiver-operator characteristic analysis gave an accuracy on a segment-by-segment analysis of 0.73 for CT, 0.87 for CTAP and 0.82 for MRI. Combining information from CT and CTAP, CT and MRI, or all three modalities, did not significantly increase the percentage of patients staged correctly (71, 72 and 76 per cent respectively). CONCLUSION: The diagnostic accuracy of spiral CT, MRI and CTAP was similar. Combining modalities did not improve accuracy.

Delayed hepatic artery thrombosis in adult orthotopic liver transplantation-a 12-year experience.

BACKGROUND: Although the clinical features of early hepatic artery thrombosis (HAT) are well defined, the features of delayed (more than 4 weeks after transplantation) hepatic artery thrombosis are less clearly defined. The aim of our study was to identify risk factors, clinical presentation, and outcome of management of delayed hepatic artery thrombosis (HAT) after liver transplant (LTx). METHODS: An analysis of prospectively collected data of all patients transplanted from 1986 to 1998 was performed. The importance of recipient (age, sex, primary indication for LTx, cytomegalovirus status, and intraabdominal sepsis) and donor factors (donor age, cold ischemia time, and donor cytomegalovirus status), modes of presentation, and outcome of treatment (biliary reconstruction/stenting, regraft, vascular reconstruction, observation) were analyzed. RESULTS: Delayed HAT was seen in 31/1097 adult LTx recipients (incidence 2.8%). No recipient or donor factors were identified as risk factors. A total of 16 patients were symptomatic at presentation (HAT diagnosed on abdominal ultrasound). Six patients had recurrent episodes of cholangitis, four had cholangitis with a stricture, four had cholangitis and intrahepatic abscesses, and two had bile leaks. Biliary reconstruction was done in six patients (all of whom subsequently required a regraft), vascular reconstruction was performed in two patients (one regrafted and one died shortly after), four patients with cholangitis and stricture on presentation had a biliary stent (all four were later regrafted). A total of 16 patients were regrafted, 9 are alive, 5 died within 6 months (septic at time of LTx), 1 died after 1 year, and 1 died after 2 years. Fifteen patients were asymptomatic and detected on routine screening. 5 have remained asymptomatic and are still alive, 1 developed a biliary stricture that was stented and is alive 105 months later, 4 had recurrence of the original disease, 3 developed progressive graft failure and were listed for transplant but died before regraft due to overwhelming sepsis and hepatic encephalopathy. Two patients died due to nonbiliary sepsis. CONCLUSIONS: Delayed HAT is a rare complication of LTx that may present with biliary sepsis, or remain asymptomatic. Biliary or vascular reconstructions do not increase graft survival. Of the patients who were clinically silent on presentation, 20% developed progressive graft failure requiring a second transplant. A total of 33% survived in the long-term without a second transplant. Ongoing severe sepsis at the time of regraft results in poor survival.

pH-induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: implications for zymogen activation.

The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N-terminal, 81-residue inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep 'autocatalytic' removal of the prodomain at the C-terminal side of the two consensus sites, R(78)-T-K-R(81) approximately and R(44)-G-V-T-K-R(49) approximately. The furin-mediated cleavage at R(44)-G-V-T-K-R(49) approximately, in particular, is significantly accelerated in an 'acidic' environment. Here, we show that under neutral pH conditions, the inhibitory prodomain of furin is partially folded and undergoes conformational exchanges as indicated by extensive broadening of the NMR spectra. Presence of many ring-current shifted methyl resonances suggests that the partially folded state of the prodomain may still possess a 'semirigid' protein core with specific packing interactions among amino acid side chains. Measurements of the hydrodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformational stability of the prodomain appears to be pH sensitive, in that the prodomain undergoes an unfolding transition towards acidic conditions. Our NMR analyses establish that the acid-induced unfolding is mainly experienced by the residues from the C-terminal half of the prodomain (residues R(44)-R(81)) that contains the two furin cleavage sites. A 38-residue peptide fragment derived from the entire pH-sensitive C-terminal region (residues R(44)-R(81)) does not exhibit any exchange-induced line broadening and adopts flexible conformations. We propose that at neutral pH, the cleavage site R(44)-G-V-T-K-R(49) approximately is buried within the protein core that is formed in part by residues from the N-terminal region, and that the cleavage site becomes exposed under acidic conditions, leading to a facile cleavage by the furin enzyme.

Percutaneous portal vein thrombolysis and endovascular stent for management of posttransplant portal venous conduit thrombosis.

BACKGROUND: Thrombosis of a portal vein conduit after liver transplant is an uncommon clinical situation. Percutaneous thrombolytic therapy for this condition has not been widely described. METHODS: We describe a case of thrombosis of a portal vein (PV) conduit subsequent to orthotopic liver transplantation that was successfully treated by percutaneous portal vein thrombolysis by using tissue plasminogen activator, angioplasty, and endovascular stent placement. RESULTS: A satisfactory outcome was achieved with a patent portal vein, on ultrasound, at 8-month follow-up. CONCLUSION: A percutaneous transhepatic approach to treatment of thrombosis of a portal vein conduit appears to be a promising technique to use to avoid surgery, with good medium-term results.

Inhibitory activity and structural characterization of a C-terminal peptide fragment derived from the prosegment of the proprotein convertase PC7.

Mammalian proprotein convertases (PCs) belong to the family of recently discovered serine proteases responsible for the processing of a large number of precursor proteins into their active forms. The enzymatic activities of the convertases have been implicated in a variety of disease states, such as cancer and infectious and inflammatory diseases. Like many other proteases, PCs are also synthesized as inactive proenzymes with N-terminal extensions as their prosegments. Here, we present the inhibitory activities of a number of "putative" interfacial peptide fragments derived from the proregion of PC7. We found that a peptide fragment corresponding to the C-terminal region (residues 81p-104p, or C24: E(1)-A-V-L-A-K-H-E-A-V-R-W-H-S-E-Q-R-L-L-K-R-A-K-R(24)) of the PC7 prosegment displays a strong inhibition (K(i) = 7 nM) of the PC7 enzyme comparable to that of the full-length (104 residue) prosegment. The same 24 residue peptide shows significantly populated helical conformations in an aqueous solution close to the physiological condition. Structure calculations driven by NOE distance restraints revealed a slightly kinked helical conformation for the entire peptide, characterized by many side-chain/side-chain interactions including those involving charged residues E8-R11-E15 and hydrophobic residues W12 and L19. These results suggest that the C-terminal region of the prosegment of PC7 may play a dominant role in conferring the inhibitory potency to the cognate enzyme and this strong inhibitory activity may be a direct consequence of the folded conformation of the peptide fragment in solution. We surmise that such a structure-function correlation for an inhibitory peptide could lead to the design and discovery of molecules mimicking the specific interactions of the PC prosegments for their cognate proteases.

Fluoroalcohols as structure modifiers in peptides and proteins: hexafluoroacetone hydrate stabilizes a helical conformation of melittin at low pH.

The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH approximately 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 M (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of approximately 8 M. Temperature dependent CD experiments support a 'cold denaturation' of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 M HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NH/NHi+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10-G11-T12. The helical structure of melittin obtained at 3.6 M HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.

Synthetic interface peptides as inactivators of multimeric enzymes: inhibitory and conformational properties of three fragments from Lactobacillus casei thymidylate synthase.

Three synthetic peptides corresponding to distinct segments of the subunit interface of the dimeric enzyme thymidylate synthase (residues 17-38, N 22; residues 174-190, M 17; and residues 201-220, C 20) have been investigated for their ability to function as inhibitors by modifying the quaternary structure of the enzyme. A dramatic reduction of enzyme activity is observed following incubation of TS with the C 20 peptide. The N 22 and M 17 peptides were unable to cause any loss of enzymatic activity. Addition of the C 20 peptide results in a loss of fluorescence of TS labeled with a dansyl group at Cys 198, following aggregation and precipitation of the protein. The effects are not observed for the N 22 or M 17 peptides. Loss of enzymatic activity is related to the ability of C 20 to promote protein aggregation. The conformations of the peptides have been studied using CD and NMR in order to correlate the observed function with solution structures. Peptides N 22 and M 17 are largely unstructured in aqueous solution. A population of nascent helical structures or multiple turn conformations has been detected for the C 20 peptide in aqueous solution by NMR. Addition of 50% (v/v) hexafluoroacetone trihydrate (HFA), a structure-stabilizing cosolvent, stabilizes the helical conformation in the C 20 peptide. Under similar conditions, N 22 and M 17 remain largely extended with observations of local beta-turn conformations. Interestingly, the C 20 peptide is a beta-hairpin in the native structure, whereas the other two peptides are individual strand components of a beta-sheet.

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.

Effects of organic solvents on protein structures: observation of a structured helical core in hen egg-white lysozyme in aqueous dimethylsulfoxide.

A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO (< 10%) have little effect on the overall folded conformation of lysozyme as seen from 1H NMR chemical shift dispersion. At increasing DMSO concentrations (> 10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by approximately 50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain-side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel beta-sheet domain are largely solvent-exposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of non-aqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins.

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

Omega amino acids in peptide design: incorporation into helices.

Incorporation of easily available achiral omega-amino acid residues into an oligopeptide results in substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic. The central Gly-Gly segment of the helical octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe(1) has been replaced by delta-amino-valeric acid (delta-Ava) residue in the newly designed peptide Boc-Leu-Aib-Val-delta-Ava-Leu-Aib-Val-OMe(2). 1H-nmr results clearly suggest that in the apolar solvent CDCl3, the delta-Ava residue is accommodated into a folded helical conformation, stabilized by successive hydrogen bonds involving the NH groups of Val(3), delta-Ava(4), and Leu(5). The delta-Ava residue must adopt a gauche-gauche-trans-gauche-gauche conformation along the central polymethylene unit of the aliphatic segment, a feature seen in an energy-minimized model conformation based on nmr parameters. The absence of hydrogen bonding functionalities, however, limits the elongation of the helix. In fact, in CDCl3, the folded conformation consists of an N-terminal helix spanning residues 1-4, followed by a Type II beta-turn at residues 5 and 6, whereas in strongly solvating media like (CD3)2SO, the unfolding of the N-terminal helix results in beta-turn conformations at Leu(1)-Aib(2). The Type II beta-turn at the Leu(5)-Aib(6) segment remains intact even in (CD3)2SO.CD comparisons of peptides 1 and 2 reveal a "nonhelical" spectrum for 2 in 2,2,2-trifluoroethanol.

Solid state and solution conformations of a helical peptide with a central Gly-Gly segment.

The influence of amino acids with contrasting conformational tendencies on the stereochemistry of oligopeptides has been investigated using an octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe, which contains two helix-promoting Aib residues and a central helix-destabilizing Gly-Gly segment. Single crystal x-ray diffraction studies reveal that a 3(10)-helix is formed up to the penultimate Aib residue, at which point there is a helix reversal in the backbone, reminiscent of a C-terminal 6-->1 hydrogen bond. The curious feature in the crystal is the solvation of the possible 6-->1 bond by a CH3OH molecule, where the OH is inserted between O(3) and N(8) and participates in hydrogen bonds with both. The cell parameters are as follows: space group P2(1)2(1)2(1), a = 10.649 (4) A, b = 15.694 (5) A, c = 30.181 (8) A, R = 6.7% for 3427 data (magnitude of F0 > 3 sigma F) observed to 0.9 A. Nuclear magnetic resonance studies in CDCl3 using NH group solvent accessibility and nuclear Overhauser effects as probes are consistent with a 3(10)-helical conformation. In contrast, in (CD3)2SO, unfolding of the central segment results in a multiple beta-turn structure, with beta-turn conformations populated at residues 1-2, 3-4, and 6-7. CD studies in methanol-2,2,2-trifluoroethanol (TFE) mixtures also provide evidence for a solvent-dependent structural transition. Helical conformations are populated in TFE, while type II beta-turn structures are favored in methanol.